BIO 1140

Topic 1 – Cells and their organelles

1.1 BIOLOGICAL LEVELS OF ORGANIZATION

 A cell is the smallest collection of matter that can be alive. The
cell is the fundamental unit of life.
 The cell theory states that:
o All living organisms are composed of one or more cells
o Cells are the basic structural unit of life
o All life comes from pre-existing cells
 The unaided eye can see objects as small as ~ 0.1mm.
 Most cells are too small to see without additional
magnification. To observe them, we use a microscope.

Many Types of Microscopes Each with a Specific

Application
 In a light microscope (LM), visible light is passed through a
specimen and then through glass lenses
 Staining can be used to enhance the details of what we can
see using light microscopy
 In fluorescence microscopy (a subtype of light microscopy)
specific molecules are visualized with fluorescently-labelled dyes, antibodies or proteins
 Allows for subcellular localization to be determined
 e.x. – nucleus (blue), mitochondria (orange), cytoskeleton (green)

 To view subcellular structures, we must use the electron microscope

o Scanning electron microscopes (SEMs) focus a beam of electrons onto surface of a
specimen, providing 3-D images
o Transmission electron microscopes (TEMs) focus a beam of electrons through a specimen,
to study internal structures

Why are cells small?

 The membrane which surrounds a cell, allows for the
passage of nutrients into and waste out of the cell
 A cell also relies in large part on the diffusion of
molecules throughout its interior for proper function

 As a cell increases in size, its volume (V) grows faster

than its surface area (SA)
 SA increases by a factor of n2 but V increases by a
factor of n3
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 Small cells have a greater surface area relative to volume allowing for effective metabolism
 Therefore, larger organisms typically have more cells rather than larger cells

All cells share four common characteristics:

1. Cell membrane - an outer covering that separates the cell’s interior from its surrounding
environment
2. Cytosol - the jelly-like filling of the cell in which other cellular components are found
3. DNA - the genetic material of the cell
4. Ribosomes - complex molecule that synthesizes proteins

Prokaryotic cells are characterized as having:

 No nucleus (pro = ‘before’, -kary = ‘nucleus’)
o DNA in an unbound region called the nucleoid
 No membrane-bound organelles
 Cytoplasm bound by plasma membrane
 Average size = 1-5 m
 Most prokaryotic cells are smaller, and have a higher surface-to-volume ratio, than
eukaryotic cells – they can exchange nutrients with the outside environment faster than
eukaryotic cells

 Earliest evidence of prokaryotic life ~ 3.5 billion years ago
o Stromalites

Eukaryotic cells are characterized as having:

 DNA in a nucleus bounded by a membranous nuclear envelope (eu = ‘true’, -kary = nucleus)
 Membrane-bound organelles
 Cytoplasm between plasma membrane and nuclear membrane
 Generally larger than prokaryotic cells, average 10-100 m

Membrane bound organelles

 Mitochondria are found in essentially all eukaryotic cells
 Chloroplasts are found in plants and algae

Evolution of Eukaryotes
• Development of internal membranes
• The endosymbiotic theory suggests that an early ancestor of
eukaryotes engulfed an aerobic (oxygen-using) non-
photosynthetic prokaryote
• The engulfed prokaryote was maintained within the host cell
and became an endosymbiont, eventually evolving into a
mitochondrion
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• A second endosymbiotic event involved a photosynthetic prokaryote being engulfed by a
eukaryote containing mitochondria
• This endosymbiont evolved into a chloroplast

Evidence for the endosymbiotic theory

 Mitochondria and chloroplasts resemble bacteria in numerous ways:
• Size  the size of a prokaryote
• Bound by two outer membranes
• Grow and reproduce independently in cells using prokaryotic-like mechanisms
• Contain their own circular DNA molecules
• Contain free ribosomes to synthesize protein

CELL PARTS CATALOGUE

Prokaryotic cell Animal cell

Plant cell

Plasma Membrane
 Found in all cells
 Location: very thin barrier, consisting of a lipid bilayer and
associated proteins, which separates the internal and
external environments of the cell
 Function
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o The semipermeable lipid bilayer, allows the passage of sufficient amounts of oxygen,
nutrients, and waste to support the cell volume
o Also plays key roles in cell communication, cell adhesion and cellular structure
The Endomembrane system
• The endomembrane system is a complex and dynamic player in the cell’s compartmental
organization
• Components of endomembrane system include:
1) Nuclear envelope
2) Endoplasmic reticulum
3) Golgi apparatus
4) Lysosomes
5) Vacuoles
6) Plasma membrane
• These components are either continuous, or are connected
indirectly via vesicles

Nucleus
 Found in all eukaryotic cells
 Location
o Within the cytoplasm, it is the most visible organelle inside of the cell
o The nuclear envelope encloses the nucleus and is a double membrane (each consists of
a distinct lipid bilayer)
o Pores regulate entry and exit of molecules from the nucleus
o The nuclear lamina, a protein matrix, helps maintains its shape
 Function
o The nucleus contains most of the cell’s genetic material
o Within the nucleus, the deoxyribonucleic acid (DNA, that contains these genes) is
organized into discrete units called chromosomes

Nucleolus
 Found in all eukaryotic cells
 Location - A visible density within the nucleus, it is NOT
membrane bound
o Its appearance differs across the cell cycle (see
image on the following slide)
o It is composed of the rRNA genes, rRNA (ribosomal
ribonucleic acid) and associated proteins
 Function - The nucleolus is the site of rRNA synthesis
and ribosomal subunit assembly

Endoplasmic Reticulum (ER)

 The ER is the biosynthetic factory of the cell.
o It is found in all eukaryotic cells.
 Location – The ER is a membranous structure found within the cytoplasm
o The ER membrane is continuous with the nuclear envelope
o ER accounts for more than half of the total membranes in many eukaryotic cells
o Its internal compartment is known as the ER lumen
 The ER has two distinct structural regions:
o Smooth ER, which lacks ribosomes
o Rough ER, surface is studded with ribosomes
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Functions of Smooth ER Endoplasmic Reticulum

Nuclear
 Synthesizes lipids Nuclear envelope Smooth Endoplasmic
pore Reticulum
 Metabolizes carbohydrates
 Detoxifies drugs and poisons
 Stores calcium ions
o Part of the ER network in a cultured mammalian cell, stained with an antibody that binds
to a protein retained in the ER. The ER extends as a network throughout the
Cisternal

entire cytosol, so that all regions of the cytosol are close tospace
some portion of the
ER membrane. Cisternae
Rough Endoplasmic
o Part of an ER network in a living plant cell that was genetically engineered Reticulum
to express a
Ribosomes

fluorescent protein in the ER

 Fun Fact – the sER can double in size when an overload of metabolic products of ethanol or
barbiturates are present (ex – following heavy drinking or a drug overdose), and then return
to its original size when the waste has been processed

Functions of Rough ER
 Has ribosomes on its outer surface, which synthesize
proteins and glycoproteins (proteins modified with
covalently linked carbohydrates)
 This includes: proteins of the endomembrane system,
proteins that will be embedded in the outer cell membrane,
and proteins to be secreted from the cell (eg. protein
hormones, enzymes)
 Produces transport vesicles, which distribute lipids and
proteins to other components of the endomembrane system
 The ER is a “membrane factory” for the cell

Ribosomes
 Ribosomes are small complexes made up of both rRNA and
protein. They are NOT membrane bound.
 Found in all cells (eukaryotic and prokaryotic).
 Location
o In the cytosol as free cytosolic ribosomes (all cells)
o On the surface of the endoplasmic reticulum as ER-bound ribosomes (eukaryotes only)
o They are also found within mitochondria and chloroplasts (eukaryotes)
 Function – Carry out protein synthesis
o This is the translation of RNA into protein
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Golgi Apparatus
 The Golgi apparatus (also called the Golgi body) is the shipping and receiving center
of the cell
 It is found in all eukaryotic cells
 Location – it is a series of flattened membranous sacs (imagine a stack of pita bread)
called cisternae found within the cytoplasm, it is generally in close proximity to the ER
 Function –
o Modifies products from the ER
o e.x. – glycosylation – adding carbohydrate groups to proteins and lipids
o Manufactures some macromolecules, mainly carbohydrates
o Sorts and packages materials into transport vesicles

Travel Through the Golgi Apparatus

 The receiving side of the Golgi apparatus is called the cis face(#2
in the diagram); the opposite side is the trans face (#4/5)
 Transport vesicles from the ER (#1) fuse with the cis face and
empty their contents into the lumen of the Golgi apparatus
 As the proteins and lipids travel through the Golgi, they are
further modified so they can be sorted and packaged accordingly
(#3, 4 or 5)

Lysosomes
 Lysosome are the digestive compartment within the cell.
o The appearance (size/number/shape) of lysosomes can be
highly variable.
 The are found all eukaryotic cells.
o Relatively few plant cells have lysosomes, more commonly,
specialized vacuoles carry out lysosome-like functions in
plant cells
 Location – membrane bound compartment within the cytoplasm
o The specific properties of this membrane allow the lysosomal compartment to be more
acidic than the rest of the cell
 Function – digestion via enzymatic hydrolysis of macromolecules (proteins, fats,
polysaccharides, and nucleic acids)
o Some cells can engulf another cell by phagocytosis; forming a food vacuole
o A lysosome fuses with the food vacuole and digests
o Lysosomes also recycle the cell’s own organelles and
macromolecules in a process called autophagy (‘auto’
= self, ‘phagy’ – eating)
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Vacuoles
 Found in all eukaryotic cells
o Plant = one large central vacuole (most of the volume of the cell)
o Animal = many smaller vacuoles
 Location
o Membrane bound structure found within the cytoplasm, they are derived from the ER
and Golgi (endomembrane system)
 Function = predominantly storage but varies by cell type
o Food vacuoles are formed by phagocytosis
o Contractile vacuoles (protists), pump excess water
out of cells
o Central vacuoles (plant cells) hold organic
compounds and
water

Peroxisomes – produced during their oxidation reactions

 Peroxisomes are specialized metabolic compartments
o their origin and relation to other organelles remains unclear
 Found in all virtually all eukaryotic cells
 Location, found within the cytoplasm, they are surrounded by a single membrane.
o A crystalloid enzyme core is apparent in some peroxisomes
 Function
o Peroxisomes break down organic molecules by the process of oxidation to produce
hydrogen peroxide
o hydrogen peroxide is in turn quickly converted to oxygen
and water
o Peroxisomes perform reactions with many different metabolic
functions
o e.x. breakdown of fatty acids and amino acids necessary
to produce other biomolecules such as cholesterol and
phospholipids

Mitochondria
 They are found in virtually all eukaryotic cells
 Location – they are a double membrane bound organelle, found within the cytoplasm
o The outer membrane is smooth and the inner membrane folds into cristae increasing its
surface area
o The inner membrane creates two aqueous compartments: intermembrane space and
the mitochondrial matrix
 Function – They carry out oxidative metabolism, a metabolic process that converts stored
energy (macromolecules) to ATP using oxygen
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Chloroplasts
 Chloroplasts are found in plants and algae
 Location – found within the cytoplasm, chloroplasts are surrounded
by two membranes
o The internal compartment is filled with an aqueous solution
called the stroma
o The internal thylakoid membranes contain chlorophyll and form
membranous sacs
o Thylakoids are interconnected and stacked to form grana
o Inside of the thylakoids is a compartment called the thylakoid lumen
 Function = Carry out photosynthesis
o a metabolic process that uses the energy from sunlight to fix carbon (from CO 2) and uses
it to generate energy-rich organic molecules such as glucose
 Thin section electron micrograph of a young tobacco chloroplast. Two envelope membranes
(EM) surround the chloroplast stroma (S), within which stacked grana thylakoids (GT) and
unstacked stroma thylakoids (ST) can be recognized. Plastoglobuli (PG) and DNA-containing
regions (arrows) are also seen.

Cytoskeleton
 The cytoskeleton is a network of protein fibers that extends throughout the cytoplasm
o All cells have a cytoskeleton (prokaryotes and eukaryotes)
 Function:
o It organizes the cell’s structures and activities, anchoring many organelles
o It helps to support the cell and maintain its shape
o The cytoskeleton interacts with motor proteins to produce cell
motility
o Inside the cell, vesicles can travel along cytoskeleton “tracks”

Centrosome
 In most animal cells, microtubules grow out from a centrosome near
the nucleus
 The centrosome is known as the “microtubule-organizing center”
o In animal cells, the centrosome consists of a pair of centrioles at
right angles to one another
o Most plant cells do not have centrioles , instead microtubules
originate from many small nucleation sites within the cytoplasm
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 Another important function of the microtubule
component of the cytoskeleton is chromosome
separation during cell division

Cilia and Flagella

 Microtubules control the beating of cilia and
flagella
o locomotor appendages which protrude from
some eukaryotic cells (e.x. – spermatozoa)
 Cilia and flagella share a common structure
o A core of microtubules encased by the plasma
membrane
o A basal body that provides an anchor
o A motor protein called dynein that drives the
bending movements
 They are different from prokaryotic flagella which
are composed of a protein called flagellin and
move in a rotary pattern

Cell Wall
 The cell wall is an extracellular
structure that distinguishes plant
cells from animal cells. Prokaryotes,
fungi, and some protists also have
cell walls.
o The prokaryotic cell wall is made up of peptidoglycan (a polymer of sugar and amino
acids)
o Plant cell walls are made of cellulose (a type of polysaccharide) embedded in other
polysaccharides and protein
 Function:
o Cell wall protects, maintains shape, and prevents excessive uptake of water in plant cell

Topic 2

Plasma Membrane
 The plasma membrane (PM) is the boundary that separates a living cell from its
surroundings
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 This is a very thin barrier ~5-10nm
 The plasma membrane exhibits selective
permeability, allowing some
substances to cross it more easily than
others
o This allows the internal and external cellular environments to differ from one another
Composition of the Plasma Membrane
 (Listed in relative order of abundance by mass)
o Proteins ~50%
o Lipids ~40%
o Phospholipids
o Cholesterol
o Carbs ~10%

Membrane composition varies

 Myelin, an outgrowth of a specialized cells' membrane
that insulates neuronal axons. It contains only 18
percent protein and 76 percent lipid.
 The mitochondrial inner membrane contains 76
percent protein and only 24 percent lipid.
 The plasma membrane of human red blood cells is 30
percent lipid.

Phospholipids
 In phospholipids (diacylglycerol), two
fatty acids and a phosphate group are attached to
glycerol
o The two fatty acid tails are hydrophobic
o The phosphate head group is hydrophilic
o The phosphate may be modified by the addition of charged
or polar chemical groups
 Therefore, we can say that they are amphipathic molecules,
containing both hydrophobic and hydrophilic regions

Fatty acids vary in length, number and location of double

bonds
 Saturated fatty acids do not have double bonds
o Solid at room temperature
 Unsaturated fatty acids have one or more double bonds
o May be liquid at room temperature
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Phospholipids are a major constituent of the plasma membrane
 When phospholipids are added to water, they spontaneously self-assemble into a bilayer, due
to their structure
o The hydrophilic head groups of the phospholipids face the aqueous solution
o The hydrophobic tails are sequestered in the middle of the bilayer
 The phospholipid bilayer exists as a stable boundary between the two aqueous compartments
 Phospholipids contribute to dynamic nature of the plasma membrane

Representing the Plasma Membrane

• Fluid Mosaic Model - Model of a typical animal cell plasma membrane

Membrane Fluidity
 Membranes are NOT static
o Phospholipids and proteins in the plasma membrane can move within the bilayer
o Most move/drift laterally, rarely a molecule flip-flops transversely (between layers of
membrane)
o For membranes to work properly they must be FLUID - think salad oil!
o Temperature had a significant effect on fluidity
o As temperatures cool, membranes go from a fluid state to a solid state
 The temperature at which a membrane solidifies depends on its lipid composition
o This is due to the effect of double bonds on phospholipid packing
o Membranes rich in unsaturated fatty acids are more fluid than those rich in
saturated fatty acids

Membrane Fluidity and Cholesterol

 The steroid lipid, cholesterol has different effects on membrane fluidity at different
temperatures
o At warm temperatures, it restrains movement of phospholipids, preventing the membrane
from becoming too fluid
o At cool temperatures, it maintains fluidity by preventing tight packing

Membrane Lipid Composition

 Variations in the lipid composition of cell membranes of many species are likely due to
adaptations to specific environmental conditions
 Further, the ability to change membrane lipid composition as temperature changes has
evolved in organisms that live where temperatures vary

Membrane Proteins
 A membrane is a mosaic of different proteins, often grouped together, embedded in the fluid
matrix of the lipid bilayer
 Proteins determine most of the membrane’s specific functions
 There are two types of membrane proteins:
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o Peripheral proteins are found on the surface of the membrane and are attached to
either integral proteins or phospholipids
o Integral proteins penetrate the hydrophobic core, and are embedded in the membrane

Integral Proteins
 Integral proteins penetrate the hydrophobic core of the phospholipid bilayer and are
embedded in the membrane
 The hydrophobic regions of an integral protein consist of one or more stretches of nonpolar
amino acids, often coiled into alpha helices
 The locations and number of hydrophobic regions determine how they arrange within the
bilayer

Transmembrane proteins
 Integral proteins do not have to cross both layers of the membrane
 Those that do span the membrane are called transmembrane proteins
 These transmembrane domains can be organized to create a hydrophilic channel through the
membrane
o e.x. – aquaporin

Membrane proteins have many functions

Example - Clinical Importance

 HIV must bind to the immune cell surface protein CD4 and a “co-receptor” CCR5 in order to
infect a cell
 HIV cannot enter the cells of resistant individuals that lack
CCR5

Carbohydrates
 Located only on the exterior surface of the plasma
membrane, covalently bound to either:
o proteins = glycoproteins, or lipids = glycolipids,
considered together = glycocalyx
 Function  cell-cell recognition and attachment, increase
hydrophilicity of the cell (↑ interaction with extracellular
environment)
 Carbohydrates of the plasma membranes vary among species, individuals, and even cell
types in an individual
o e.x.– blood type reflects the variation in the
carbohydrate component of the glycoprotein on the
surface of red blood cells

Membrane Sidedness
 Plasma membranes are asymmetric; the inner surface differs from the outer surface
o Ex. Interior proteins anchor fibers of the cytoskeleton to the membrane
o Exterior proteins bind to the extracellular matrix
o Glycoproteins and glycolipids are only on the outer leaflet
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o Different phospholipid composition
o outer leaflet has more phosphatidylcholine
and sphingomyelin,
o inner leaflet has more phosphatidylethanolamine,
phosphatidylserine and inositol containing
phospholipids

Membrane synthesis
 The asymmetric distribution of plasma membrane
components is determined when the membrane is synthesized and assembled by the ER and
Golgi apparatus

Travel Through the Endomembrane System

 Lipids and proteins are synthesized in the endoplasmic reticulum (ER), proteins may acquire
carbohydrates here
 Vesicles travel from the ER to the cis-face of the Golgi apparatus where they fuse
 In the Golgi, lipids may acquire carbohydrates and proteins may be further modified
 Vesicles with membrane proteins, secretory proteins and glycolipids bud off of the trans-face
of the Golgi
 Vesicles travel to the plasma membrane where they fuse releasing their contents into the
extracellular space
 The internal layer of the vesicle’s lipid bilayer is now on the outer surface of the cell
membrane and the vesicle’s outer lipid layer is in the internal leaflet of the plasma membrane

TRANSPORT ACROSS CELL MEMBRANES

 Plasma membranes are selectively permeable, regulating the cell’s molecular traffic
o Hydrophobic (nonpolar) molecules can dissolve in the lipidbilayer and pass through the
membrane readily
o Hydrophilic (polar) molecules do not cross the
membrane easily
o Various transport proteins allow hydrophilic
substances to pass across membrane

Diffusion
 Diffusion is the tendency for molecules to spread out
evenly into the available space
 This is a spontaneous process driven by the thermal energy of the molecules involved
 Each molecule moves randomly but substances diffuse down their concentration
gradient (high conc  low conc)
 Diffusion of a substance across a biological membrane is also known as passive transport
o no energy is expended by the cell to make it happen
 Only small nonpolar molecules can diffuse readily through biological membranes (e.x. - O 2,
CO2, & steroid hormones)

Factors Affecting Diffusion

 Concentration gradients - Greater difference,
faster diffusion
 Mass of the molecules - Smaller molecules diffuse
more quickly
 Temperature - Molecules move faster when
temperatures are higher
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 Solvent density - Dehydration increases density of cytoplasm – reduces diffusion rates
 Solubility – more nonpolar (lipid-soluble) materials diffuse faster
 Surface area – increased surface area speeds up diffusion rates
 Distance travelled – the greater the distance, the slower the rates; important factor
affecting upper limit of cell size
 Pressure – in some cells (i.e. kidney cells), blood pressure forces solutions through
membranes, speeding up diffusion rates

Facilitated Diffusion
 Facilitated transport (a.k.a. facilitated diffusion) moves substances down their concentration
gradients through integral membrane proteins
o Ions and small polar molecules diffuse across the membrane this way
 There are two types of facilitated transport proteins:
o Channel proteins
o Carrier proteins

Channel proteins
 Channel proteins provide corridors that allow a specific molecule or ion to cross the plasma
membrane
o Some are open all the time
o Others are gated and only open when a specific signal is received
 Important examples:
o Aquaporins - specific to H2O
o Muscle cells have gated ion channels allowing muscle contraction when opened

Carrier proteins
 Carrier proteins are specific to a particular substance
 They bind to that substance, change conformation (shape) & translocate or “carry” it to the
other side.
 Many allow movement in either direction, as concentration gradients change
o Important example - Glucose transport proteins (GLUTS)

How water crosses the membrane

 Even though it is polar, water can diffuse passively across the plasma membrane
 This isn’t an easy journey
 Water can also travel across the plasma membrane though aquaporin channels

Diffusion of Water – A Special Case

 Osmosis is the diffusion of water across a membrane
 Differences in water concentration occur when a solute cannot pass through the selectively
permeable membrane
 When this occurs, “free” water diffuses across the membrane from the region of lower solute
concentration to the region of higher solute concentration until the solute concentration is
equal on both sides

Water balance of cells

 Tonicity refers to the ability of a surrounding solution to
cause cells to gain or lose water
 Isotonic solution: solute concentration is the same as
that inside the cell  no net water movement across the
plasma membrane
 Hypertonic solution: solute concentration is greater than that inside the cell  cell loses
water
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 Hypotonic solution: solute concentration is less than that inside the cell  cell gains water
 Animal cells function best when extracellular fluids are isotonic
Water Balance in Plant Cells
 The cell walls help maintain water balance
 Plant cell in hypotonic solution swells until wall opposes uptake; it is turgid (firm)
 Plant cell in isotonic environment, no net movement of water; it is flaccid (limp)
 In a hypertonic environment, plant cells lose water, and plasma
membrane pulls away from the cell wall causing plant to wilt;
plasmolysis (usually lethal)
 Without adequate water, the plant on the left has lost turgor
pressure, visible in its wilting; the turgor pressure is restored by
watering it (right).

Osmoregulation
 Hypertonic or hypotonic environments create osmotic problems for organisms
 Osmoregulation, the control of solute concentrations and water balance, is a necessary
adaptation for life in such environments
 Freshwater protists, like paramecia and amoebas, use contractile vacuoles, to pump water
out of their cells so they do not burst since they are hypertonic to their environment

ACTIVE TRANSPORT uses energy, usually in the form of

ATP, to move a substance against: its concentration gradient
o i.e. from low to high concentration
OR
o its electrochemical gradient
o ex. moving H+ ions to a solution that is already more positively charged

Carrier proteins
 Active transport occurs through transmembrane, carrier proteins
called pumps.
o Uniporter carries one molecule or ion
o Symporter carries two different molecules or ions, in the
same direction
o Antiporter carries two different molecules or ions, in
different directions
 Active transport allows cells to maintain gradients that differ from their surroundings.
 An electrical gradient, where the cytoplasm contains more negatively charged molecules
(more neg ions & proteins) than the extracellular fluid, is critical for proper cell functioning.
 The sodium-potassium pump (Na+-K+ pump) is an important example of an active
transport system.
o It moves 3 Na+ out and 2 K+ into the cell using 1 ATP

Questions:
 Is this pump a uniporter, symporter or antiporter?
o ANTIPORTER
 This pump is an electrogenic pump. What do you think that means?
o HELPS MAINTAIN POSITIVE AND NEGATIVE CHARGES ON EITHER SIDE OF THE CELL
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Cotransport: Coupled Transport by a Membrane Protein

(secondary active transport co-transport)
 Cotransport occurs when active transport of a solute indirectly
drives transport of other solutes
 Plants commonly use a hydrogen ion gradient to drive active
transport of nutrients into the cell

Example - Clinical Importance

 Treatment of diarrhea - a serious, potentially lethal problem in some parts of the world.
 WHY?
o Normally, sodium in waste is reabsorbed in the colon, maintaining constant levels in the
body.
o Diarrhea expels waste so rapidly that reabsorption is not possible, and sodium levels fall
precipitously.
 Treatment - patients are given a solution to drink containing high concentrations of salt
(NaCl) and glucose. The solutes are taken up by sodium-glucose cotransporters on the
surface of intestinal cells and passed through the cells into the blood.

Bulk transport
 Sometimes cells need to import or export molecules/particles (such as polysaccharides and
proteins) that are too large to pass through a transport protein
o Bulk transport is another type of active transport (energy is required)
o Bulk export is called exocytosis (‘exo’ = out)
o Bulk import is called endocytosis (‘endo’ = in), there are 3
subtypes:
 Phagocytosis (“cellular eating”)
 Pinocytosis (“cellular drinking”)
 Receptor-mediated endocytosis

Exocytosis
 In exocytosis, transport vesicles migrate to the membrane, fuse with it, and release their
contents to the outside of the cell
 Examples
o Secretory cells use exocytosis to release their products
o Neurons release neurotransmitters to communicate with
neighboring cells
o Pancreatic cells releases insulin
o Exocytosis is used by plant cells to deliver proteins and
carbohydrates from the Golgi to the
outside of the cell for cell wall formation

Phagocytosis
 During phagocytosis a cell engulfs a
particle to form a membrane bound vacuole
 Vacuoles fuse with lysosomes to digest the
particle
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 Example – White blood cell phagocytosing a bacteria to protect the
body from it
pinocytosis
 In pinocytosis, extracellular fluid is “gulped” into tiny membrane
bound vesicles
 Internalized vesicles fuse with lysosomes to digest its contents
 Example – Important part of the surveillance of the environment
by cells of the immune system

Receptor-Mediated Endocytosis
 In receptor-mediated endocytosis, binding of ligands
(specific molecules) to receptors embedded in the
membrane triggers vesicle formation/internalization
 Example Human cells use receptor-mediated endocytosis
to take in cholesterol for membrane synthesis and the
production of
steroid
hormones

Topic 3 The Cytoskeleton and the Extracellular Matrix

The Cytoskeleton
 The cytoskeleton is a network of protein fibers extending throughout the cytoplasm
 It is composed of 3 types of fibers, each of varying size and function:
o Microfilaments
o Microtubules
o Intermediate filaments
 Generally, they act together to:
o Organize the cells’ structure
o Anchor the organelles
o Facilitate cellular activities
 The 3 Components of the Cytoskeleton - The three polymer systems of a fibroblast cytoskele-
ton, as seen in the fluorescence microscope after fixation and labelling with specific probes.
o (left, red) the actin cytoskeleton, labelled with fluorescent phalloidin;
o (middle, blue) the microtubule cytoskeleton labelled with an antibody to tubulin;
o (right, green) the intermediate filament cytoskeleton labelled with antibodies to the
intermediate filament protein, vimentin.
 Cytoskeleton is dynamic
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Microtubules
 Microtubules are hollow rods about 25 nm in diameter
 They are made up of tubulin
o Tubulin itself is a protein dimer (a protein made up of 2 polypeptide
chains)
 Functions:
o Shaping the cell (resist compression)
o Guiding movement of organelles and vesicles
o Separating chromosomes during cell division

Microtubule Dynamics
 Microtubules grow/shrink though the addition/removal of tubulin dimers
at either end of the polymer
 Microtubules are asymmetrical, with one end (the plus end) allowing
for addition/removal of tubulin at a
significantly faster rate than the other (the
minus end)

Microtubules and Centrosomes

 The minus end of the microtubule is generally
anchored to the microtubule organizing
centers (MTOCs)
o The primary MTOC in an animal cell is
called the centrosome, and it is usually
located adjacent to the nucleus
o In animal cells, the centrosome has a pair
of centrioles, each with nine triplets of
microtubules arranged in a ring
o Other eukaryotic cells organize
microtubules by other means

Microtubules and Cell Motility

 Cell motility refers to both:
o Changes in cell location (e.x. – swimming sperm)
o Movement of cell parts (e.x. movement of a vesicles within the
cell)
 This involves the interaction between the cytoskeleton and motor-
proteins which convert chemical energy to mechanical energy
o e.x – A vesicle ‘walking’ along the cytoskeleton to its destination
o e.x. - Invagination of the plasma membrane during phagocytosis

Motor proteins
 Kinesins move cargo towards the plus end of the
microtubules
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 Dynein moves cargo towards the minus end of microtubules.
o Both require ATP in order to carry out their function

Intermediate Filaments
 Intermediate filaments range in diameter from 8–12 nm and are
composed of different proteins
o e.x. - keratin, which organizes into cables
 They are one of the more permanent structures of the cytoskeleton
o Even after the death of a cell, the intermediate filament network persists
o e.x – outer layer of dead skin cells is full of keratin
 They support cell shape and fix organelles in place
o e.x. - the nucleus sits within a ‘cage’ of intermediate filaments known as the nuclear
lamina

Microfilaments
• Microfilaments are solid rods about 7 nm in diameter, built as a twisted double chain of
actin subunits
• Their structural role is to bear tension, resisting pulling forces within the cell
• It often forms a 3-D network called the cortex just inside the plasma membrane but can be
organized in a variety of ways to support different cell shapes
o e.x. - make up the core of intestinal microvilli

Microfilaments and Cell Motility

 Microfilaments that function in cellular motility contain the protein
myosin in addition to actin, allowing for various types of
movement.
 Examples -
o Contraction in muscle cells
o Ameboid movement of cells
o The circular flow of cytoplasm in plant cells known as
cytoplasmic streaming

Extracellular Structures and the Connection Between

Cells
 Cells in animals and plants are organized into tissues, organs and
organ systems
 Most cells synthesize and secrete materials external to the plasma
membrane to permit this organization while also maintaining
means of communicating with one another
 These extracellular structures include:
o Cell walls of plants
o Extracellular matrix (ECM) of animal cells
o Intercellular junctions
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Plant Cell Wall
 Functions of the plant cell wall include:
o Maintaining the shape of the cell
o Preventing excessive water uptake
o Acting as a barrier to infection
 Plant cell walls are made of cellulose embedded in other polysaccharides and protein

 Plant cell walls vary in composition and thickness

 They have multiple layers:
o Primary cell wall
o Fairly thin and flexible
o Middle lamella
o Thin layer of pectin (sticky polysaccharide) which glue
adjacent cells together
o Secondary cell wall (in some cells) (in trees!)
o Several laminated layers of strong durable protection and
support, added between the plasma membrane and the
primary cell wall when a cell stops growing

Intercellular Junctions
 Intercellular junctions provide direct channels of
communication between cells
• Plants – plasmodesmata
o channels between the cell walls of adjacent plant
cells which connect their cytoplasm and allow
materials to move from cell to cell

Communication Across the Cell Wall

 Plasmodesmata are channels between the cell walls of
adjacent plant cells which connect their cytoplasm and allow
materials to move from cell to cell

Extracellular Structures in Animal Cells

 Animal cells lack cell walls but have an elaborate extracellular matrix (ECM)
 Functions of the ECM include:
o Support
o Adhesion
o Movement
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o Regulation
Components off the ECM
 The ECM is made up of :
o Fibrous glycoproteins such as collagen and
fibronectin
o Carbohydrate rich glycoproteins attached to a
carbohydrate backbone, called proteoglycans
 Cells attach to the ECM by integrin proteins
o Integrins can transmit information between the ECM
and the cytoskeleton, integrating what is happening
both inside and outside of the cell

Intercellular Junctions
 Intercellular junctions provide direct channels of communication between
cells
o Plants – plasmodesmata
o In animal cells there are 3 main types of junctions:
o Tight Junctions
o Desmosomes
o Gap Junctions

Tight Junctions
 Tight junctions are watertight seals between animal cells that prevent
materials from leaking between cells
o The PMs are pressed rightly against each other and bound together by proteins
 They are often found between they epithelial cells that line internal organs and cavities
o e.x. – tight junctions make blood vessels watertight
Desmosomes
 Desmosomes are anchoring junctions that form when cadherins (a type of transmembrane
protein) in adjacent cells interact
o Think of them as a spot weld or rivet
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 Desmosomes are tied into the cytoskeleton by their interaction
with intermediate filaments
 These join adjacent cells in tissues that stretch (eg. heart, lungs,
muscles)

Gap Junctions
 6 connexins proteins assemble to form a channel through the plasma membrane
called a connexon or hemichannel
 When the connexons of adjacent cells are aligned, they can dock creating a channel
between the cells called a gap junction
 Hemichannels and gap junctions allow ions, and
other small signaling molecules to travel through
them

Topic 4: Metabolism
Energy is the ability to do work.
 Energy can be classified as kinetic or potential.
o Objects in motion have kinetic energy
o Thermal energy is a type of kinetic energy associated with random movement of
atoms or molecules
o Objects that have the potential to move have potential energy
o Chemical energy is a type of potential energy which is available for release in a
chemical reaction

Energy can be converted from one form to another

 The potential energy stored in the chemical bonds of gasoline can be transformed into
kinetic energy that allows a car to race on a racetrack.
All living organisms share several key characteristics or functions:
 Order
 Adapt/Evolve
 Grow and develop
 Homeostasis
 Respond to the environment
 Process energy
 Reproduce

 Bioenergetics is the study of energy flow through a living system.

 The energy that sustains most of the earth’s life forms comes from the sun.
 Example –
o Energy from the sun is captured during photosynthesis and used to convert CO2 and
H2O into glucose.
o The energy stored in glucose is later released during cellular respiration, regenerating
CO2 and H2O.

Cells Need Energy to do Work - The cell does three main kinds of work:
 CHEMICAL WORK, the pushing of energy consuming (endergonic) reactions that would not
occur spontaneously, such as the synthesis of polymers from monomers
BIO 1140
 TRANSPORT WORK, the pumping of substances across
membranes against the direction of spontaneous
movement
 MECHANICAL WORK, such as the beating of cilia, the
contraction of muscle cells, and the movement of
chromosomes during cellular reproduction
 Metabolism refers to all of the chemical reactions of
a cell or organism. Very generally, metabolism
manages the material and energy resources of the
cell.
 Metabolism is composed of many metabolic
pathways.
 A metabolic pathway is a series of biochemical
reactions that converts one or more substrates
into a final product.
o Each step is catalyzed by a specific enzyme
 Metabolic pathways are divided into two types, catabolic and anabolic, that work together
to maintain the cell’s overall energy balance.
 Metabolic reactions that …
o … require energy and synthesize larger molecules are called anabolic.
o … release energy and break down large molecules into smaller molecules are called
catabolic.

Thermodynamics is the study of energy transformations

 The laws of thermodynamics govern energy transformations.
o The first law states that the total amount of energy in the universe if
constant: energy cannot be created or destroyed.
o Plants are energy transformers NOT energy producers
o The second law states that every energy
transformation increases in the entropy (measure
of disorder) of the universe.
o Even if a reaction is generating order (e.x – building
a macromolecule), some energy is lost increasing
the disorder of the larger system.

Thermodynamics and Metabolism

 The study of thermodynamics can occur in:
o An isolated system which is unable to exchange energy or matter with its surroundings
o e.x. - liquid in a ‘perfect’ thermos
o An isolated system eventually reaches equilibrium, and can then
do no further work
o e.x. - a closed hydro electric dam or an individual metabolic
reaction occurring in a test tube
 The study of thermodynamics can occur in:
o An open system, where energy and matter can be transferred
between the system and its surroundings
o Organisms are open systems !!!
o An open system never reaches equilibrium, and can continually do
work
o Cells are not in equilibrium; they are open systems
experiencing a constant flow of energy and matter
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 e.x. - Catabolic pathway in a cell release free energy in a series of reactions whose
products are the reactants for the subsequent reaction maintaining the flow of
matter and energy
 Biologists want to know which reactions will occur without energy input (spontaneous) and
which require input of energy
o To do so, we need to determine the energy changes that occur in these chemical
reactions

Free Energy and Biological Systems

 Gibb’s Free Energy (G) = amount of energy available to do work in a system when the
temperature and pressure are uniform
o The concept of free energy can be applied to the chemistry of life’s processes
 Based on their changes in free energy, all chemical reactions, including metabolic processes
can be classified as endergonic or exergonic

Endergonic Reactions
 If a chemical reaction requires an input of
energy, then ΔG>0
o Products of these reactions will have more free energy than
the substrates
o These reactions are NOT spontaneous
o Example - Photosynthesis
o 6CO2 + 6H2O → C6H12O6 + 6O2
ΔG = 2870 kJ/mol

 What provides the energy for a cell’s endergonic

reactions?
o Usually hydrolysis of ATP (adenosine triphosphate)

Hydrolysis of ATP is Exergonic

 Bonds between the phosphate groups of ATP can be broken by
hydrolysis
o Energy is released when the terminal phosphate bond of ATP is
broken
o The release of energy comes from the chemical change to a state
of lower energy, not from the phosphate bonds themselves

ATP Hydrolysis Performs Cellular Work

 How - In the cell, the energy from the exergonic reaction of ATP hydrolysis can be used to
drive an endergonic reaction
o This phenomena is known as energy coupling
 Overall, the coupled reactions are exergonic
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An Example of Energy Coupling
 ATP Can be Regenerated
o ATP is a renewable resource regenerated from
adenosine diphosphate (ADP)
o Energy to phosphorylate ADP comes from catabolic
reactions in the
cell

Exergonic Reactions
 If energy is released in a chemical reaction, then ΔG<0
o Products of these reactions will have less free energy than the substrates
o These reactions ARE spontaneous
 Example - Cellular Respiration

ΔG= −2870 kJ/mol

C6H12O6 + 6O2 → 6CO2 + 6H2O

 Exergonic reactions are spontaneous reactions because they

can occur without the addition of energy.
 Remember - spontaneous reactions do not necessarily occur
quickly!
o Example - the rusting of iron happens slowly despite being
a spontaneous, exergonic reaction

 Activation energy (EA) is the energy required for a reaction to

proceed (the “hump” in the diagram).
 Activation energy is often supplied in the form of thermal
energy that reactant molecules absorb from their surroundings.

How does activation energy work?

 EA causes the reactant(s) to become unstable, which allows the
necessary bond(s) to be broken or made more easily.
 This unstable state is called the transition state.
 Once in the transition state, the reaction occurs very quickly.
 Consider the following reaction:
AB + CD → AC + BD
Reactants Products
BIO 1140

Catalyzed Reactions
 A catalyst is a chemical agent that speeds up a
reaction without being consumed
 An enzyme is a catalytic protein which accelerates a biochemical
reaction without being consumed
 N.B - while the overwhelming majority of biological enzymes are
proteins, some non-protein enzymes exist, including ribozymes
o Ex. The hydrolysis of sucrose by sucrase is an enzyme-
catalyzed reaction

How do enzymes catalyze reactions?

 Enzymes catalyze reactions by lowering EA barrier
 Enzymes DO NOT affect the overall change in free energy (∆G)
o Rather, enzymes speed up reactions that would occur eventually

How do enzymes lower activation energy?

 The enzyme can help the substrate reach its transition state in many ways, including:
o position two substrates so they align perfectly for the reaction
o provide an optimal environment, i.e. acidic or polar, within the active site for the reaction
o contort/stress the substrate so it is less stable and more likely to react
o temporarily react with the substrate (chemically change it) making the substrate less
stable and more likely to react.

Specificity of Enzymes
 The reactant (aka substrate) that an enzyme binds to, and therefore the reaction catalyzed by
each enzyme, is very specific. The 3D shape of the enzyme and substrate dictate this
specificity.
 Substrate molecules interact at the enzyme’s active site.
 At the active site, there is a mild shift in shape that optimizes reactions. This is called
induced fit.
 The slight changes at the active site
maximizes the catalysis.

Protein Structure Revisited

 Remember, the 3-D shape of a protein is
determined by the amino acid sequence of
the polypeptide
 The amino acids of the active site are
particularly important for the enzyme’s
function – allow binding with unique substrate(s)
 The cellular environment is also important for
enzyme function:
o Suboptimal temperatures can denature the enzyme
(loss of shape)
o Suboptimal pHs can reduce substrate-enzyme binding

Interaction at the Active Site and Induced Fit

Between an Enzyme and its Substrate
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Enzymes are NOT consumed in a chemical reaction
 After catalyzing a reaction, the product is released, and the enzyme
becomes available to catalyze another reaction
 Therefore, very small amounts of enzyme can have a huge metabolic
impact

Regulating Metabolism
 Chemical chaos would result if a cell’s metabolic pathways were not
tightly regulated
 A cell regulates its metabolism by:
o switching on or off genes that encode specific enzymes
o we will revisit this in the final section of the course
o regulating enzymatic activity

Regulating Enzyme Activity

 Enzymes can be regulated by:
o Modifications to temperature and/or pH
o Localization of the enzymes
o Availability of coenzymes or cofactors
o Production of molecules that inhibit or promote enzyme function

Cofactors and Coenzymes

 Some enzymes require one or more cofactors or coenzymes to function
o Cofactors are inorganic ions, i.e. Fe++, Mg++, Zn++
o e.x. DNA polymerase requires Zn++
o Coenzymes are organic molecules, including ATP, NADH, and vitamins
 These molecules are provided primarily from the diet.

Enzyme Inhibition
 Competitive inhibitors have a similar shape to the substrate and compete with the substrate
for the active site
 Noncompetitive inhibitors bind to the enzyme at a different location, changing the
conformation of the active site and thereby changing the
reaction rate

Allosteric Regulation
 Allosteric inhibitors bind at a site other than the active
site, reducing their activity
 Note – all non-competitive inhibition is allosteric, but not all
allosteric inhibition is non-competitive inhibition
 Allosteric activators modify the active site of the enzyme
so that the affinity for the substrate increases

Feedback Inhibition
 Feedback inhibition is where the product
of a pathway inhibits an upstream step
 e.x. ATP is an allosteric inhibitor for some
enzymes involved in cellular respiration
 e.x. Isoleucine is an allosteric inhibitor of
the first enzyme in its own biosynthetic
pathway (depicted in the figure)
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Specific Localization of Enzymes
 Structures within the cell help bring order to metabolic pathways
 Some enzymes act as structural components of membranes and/or
reside in specific organelles
 e.x. - enzymes for cellular respiration are located in mitochondria
Energy Flow and Chemical Recycling in Ecosystems
 Life is Work!
 Energy flows into an ecosystem in the form of sunlight and
ultimately leaves as heat
 Photosynthesis generates organic molecules and O2, which are used in cellular
respiration
 Cells use chemical energy stored in organic molecules to generate ATP, which powers cellular
work

 Glycolysis and the citric acid cycle are major intersections to many catabolic
and anabolic metabolic pathways

Cellular Respiration
C6H12O6 + 6 O2 6 CO2 + 6 H2O + energy
ΔG= −2870 kJ/mol
 Some of this energy is harvested from glucose in the form of ATP, across
three metabolic stages:
o Glycolysis
o Oxidation of Pyruvate and the Citric
Acid Cycle
o Oxidative Phosphorylation

Compartmentalization of Cellular
Respiration

Cellular Respiration Overview

 As we work through the metabolic pathways of cellular
respiration, notice the close link between the transfer of
energy and the movement of electrons
*remember electrons have high energy levels
 This incremental transfer of electrons allows the cell
to transfer food energy in small packages that can be
captured in the phosphate bonds of ATP

Redox Reactions
 Oxidation-reduction (redox) reactions transfer electrons

 During cellular respiration, the fuel (such as glucose) is oxidized,

and O2 is ultimately reduced

NAD - an Electron Carrier

 NAD (nicotinamide adenine dinucleotide) is an electron carrier
o This is because NAD+ accepts electrons from redox reactions while NADH donates them
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 As electrons are harvested from organic compounds (often with their proton H), they are
usually first transferred to NAD+ generating NADH

Glycolysis = sugar splitting

 During glycolysis a 6-carbon glucose ring is split into 2 pyruvate molecules, each with 3
carbons

 Glycolysis occurs in the cytosol and does not need O2

o Nearly all organisms perform glycolysis
 It consists of 10 enzymatic reactions which are divided into two major phases (6 steps):
o Energy investment phase
o Energy payoff phase

Glycolysis – Energy Investment Phase

 The first half of glycolysis involves 5 enzymes and uses two ATP molecules
 Glucose is phosphorylated twice and then split into two three-carbon molecules, called
glyceraldehyde-3-phosphate (G3P).

Glycolysis – Energy Payoff Phase

 The second half of glycolysis involves phosphorylation without ATP
investment (step 6) and produces two NADH and four ATP
molecules to generate 2 pyruvate molecules.

Glycolysis - Summary
 The net ATP production of glycolysis is 2 ATP
o These are the only ATP produced in glucose catabolism by
anaerobic cells
 Glycolysis also produced 2 NADH

Pyruvate Oxidation
 In the presence of O2, pyruvate enters the mitochondrion (in
eukaryotic cells) where oxidation continues
 Each pyruvate is oxidized to acetyl-CoA
 Pyruvate oxidation links glycolysis with the citric acid cycle and
involves 3 reactions:

The Citric Acid Cycle (CAC)

 The CAC consist of a series of 8 enzymatic reactions:
o First, the acetyl group (2C) from acetyl CoA enters the cycle
and is combined with the oxaloacetate (4C), to form citrate
(6C)
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The next seven steps decompose citrate back to oxaloacetate, making the process a
o
cycle
o The cycle runs continuously in the presence of sufficient reactants
 With each turn* of this cycle:
o citrate is oxidized, producing 3 NADH & 1 FADH 2
o 2 CO2 are released
o 1 ATP is produced
*Remember for each initial glucose molecule there will be 2 turns of the CAC doubling this output

Summary – Glycolysis, Pyruvate Ox and CAC

 At this point, glucose is completely oxidized, meaning all possible electrons have been
removed to yield:
o 4 ATP (2 from glycolysis, 2 from CAC)
o 6 CO2 (2 from oxidation of pyruvate, 4 from CAC)
o 10 NADH (2 from glycolysis, 2 from oxidation of pyruvate,
6 from CAC)
o 2 FADH2 (from CAC)

 The ATP formed to this point, have been generated by substrate-level phosphorylation –
enzyme mediated
 The remainder (and bulk) of the ATP generated during cellular respiration will be produced by
oxidative phosphorylation
o This is the process through which NADH and FADH 2 donate electrons to the electron
transport chain to power ATP synthesis

Oxidative Phosphorylation
 This is the only part of cellular respiration where O 2 is
an input
 The pathway consists of: 1) ETC and 2)chemiosmosis

 The ETC consists of a series of electron transporters embedded in the inner mitochondrial
membrane
 It breaks the large free-energy drop from NADH and FADH 2 to O2 into smaller steps that
release energy in manageable amounts
 The electron transport chain (ETC) does not generate ATP directly
BIO 1140
 Electrons from NADH and FADH2 are shuttled through a series of increasingly
electronegative complexes with O2 acting as the final electron acceptor
 In the process, protons (H+) are pumped from the mitochondrial matrix to the
intermembrane space, generating a concentration gradient across the
membrane
o This gradient is also referred to as the proton-motive force, emphasizing
its capacity to do work
Oxidative Phosphorylation – Chemiosmosis
 ATP synthase uses the exergonic flow of H+ back down its concentration
gradient to drive phosphorylation of ADP to form ATP
 This process is called chemiosmosis, the use of energy in a H+ gradient to
drive cellular work
 The total number of ATP generated by cellular respiration is ~ 30-36 per glucose
o Varies by species and how efficiently NADH from glycolysis enters
mitochondria
 Cellular respiration stores ~ 34% of the energy from glucose in ATP
C6H12O6 + 6 O2 6 CO2 + 6 H2O + energy
ΔG= −2870 kJ/mol = ATP and heat
 During cellular respiration, most energy flows in this sequence:
 glucose  NADH  electron transport chain  proton-motive force  ATP

Regulation of Cellular Respiration

 Feedback inhibition is the most common control mechanism
o If ATP concentration begins to drop, respiration speeds
up
o When there is plenty of ATP, respiration slows down
 Control is achieved by regulating key enzymes in the
pathway
o Ex - phosphofructokinase in glycolysis
 There are many other regulatory mechanisms involved in
the control of cellular respiration, including:
o e.g. - Hormonal control of glucose entry into the cell
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Aerobic vs Anerobic
 Glycolysis occurs in both aerobic and anaerobic environments.
o NAD+ is an input of glycolysis and is regenerated during oxidative phosphorylation when
O2 is present.
o Without O2, the electron transport chain will cease to operate
 Fermentation enables glycolysis to continue to make ATP by substrate-level phosphorylation
in the absence of oxidative phosphorylation driven by
the ETC
o Without fermentation, glycolysis would halt
Fermentation

Fermentation
 There are two common types of fermentation
o Lactic acid fermentation
o Occurs in muscle cells when O2 is limited,
mammalian red blood cells, & some bacteria
(ex. those in yogurt)
o Alcohol fermentation
o anaerobic yeast species (ex. – exploited in beer making)
 Much less efficient – makes only 2ATP per glucose (glycolysis)

Three Stages of Cell Signalling – An Overview

 Reception: the target cell detects a signalling molecule that binds to a receptor (often at the
cell surface as depicted here)
 Transduction: reception of signal causes receptor to initiate a signal transduction pathway
(usually a series of steps)
 Response: the transduced signal triggers a specific response in the target cell

Types of Communication

Direct Signalling
 Direct or juxtacrine signalling may be achieved through 1) channels
between adjacent cells or 2) direct physical contact with other cells
or the extracellular matrix (ECM)
 This type of signalling is particularly important during developmental
processes and immune responses
BIO 1140

Indirect Cell Signaling

 In indirect cell signalling, the signalling molecules are secreted into the cellular environment
and will only affect those cells which express the appropriate receptor
o This is much like a radio broadcast, you must have a receiver to hear it
 The signalling molecules act as a ligand, specifically binding to receptors based on their
matching conformations

Indirect Local Cell Signalling

 Autocrine signalling - A cell secretes a signalling molecule which then acts on its own
receptors
 Paracrine signalling - A cell secretes a signalling molecule which acts at receptors on
nearby cells

 Importance – growth factors use autocrine and paracrine signalling to

coordinate their effects on the cells within their local environment
 Downside – cancer cells often exploit these signalling mechanisms to
override normal controls, stimulating cell division and cell survival
allowing for tumor formation
 Synaptic communication is a specific form of paracrine signalling
which occurs at a structure called a synapse
 A synapse is the small gap between a neuron and the cell it is
communicating with
o Upon the arrival of an electrical message (action potential) at the
endfoot of the neuron, neurotransmitters are released into the
synapse and are received by receptors on the postsynaptic cell

Indirect Long Distance Cell Signaling

 Endocrine Communication
o In long-distance signalling, plants and animals use chemicals
messengers called hormones
o In animals, hormones travel via the circulatory system
o Plant hormones sometimes travel in vessels but more often
reach their targets by moving through cells or by diffusing
through the air as a gas
o The ability of a cell to respond to a hormone depends on whether or not it has the
appropriate receptor

Types Signals
 Messages received by cells can take many forms including things such as light (received by
photoreceptors – such as in our eyes) or touch (received by mechanoreceptors – such as in
our skin or the hair cells of our ears).
 Far more often the signals received by cells are chemical in nature
o These chemical signalling molecules will therefore be the
focus of our unit on cell signalling

Types of Chemical Messengers

 Chemical messengers can take many forms:
o Peptides and proteins - e.x. oxytocin and insulin
o Amino acids - e.x. glutamate (Glu)
o Nucleotides - e.x. ATP
o Steroids - e.x. cortisol
o Retinoids - e.x. vitamin A
BIO 1140
o Lipids and lipid derivatives e.x. - IP3 and DAG
o Gases such e.x. - nitric oxide (NO)

Types of Receptors
 Most hydrophilic signalling molecules bind to cell-surface
receptor proteins that span the plasma membrane
 Small or hydrophobic signalling molecules can readily cross the
membrane, to bind and activate intracellular receptors
o These receptors are found either in the cytosol or the
nucleus of target cells

Intracellular Receptors
 Dissolved gasses and small hydrophobic molecules can
diffuse freely across the plasma membrane into the cell
o e.x. – nitric oxide (NO), steroid hormones, vitamin D, thyroid
hormones and retinoids
 Once inside of the cell these molecules interact with intracellular receptors
o e.x. - an activated hormone-receptor complex can act as transcription factor
 Example of signalling via an intracellular receptor - Aldosterone
o Secreted by the adrenal glands, aldosterone affects only kidney cells
o With aldosterone attached, the active form of the receptor protein enters the nucleus
where it acts as a transcription factor
o This aldosterone-receptor complex turns on expression of specific genes that control
water and sodium flow in kidney cells, ultimately affecting blood volume

Cell-Surface Receptors
 The three largest classes of cell-surface receptor proteins are:
o Ion channel linked receptors
o G protein coupled receptors
o Enzyme-linked receptors

 Although cell-surface receptors represent 30% of all human proteins, determining their
structures has proven challenging
o cell-surface receptors tend to be flexible and inherently unstable, thus difficult to
crystallize for analysis using X-ray crystallography

G Protein Coupled Receptors (GPCRs)

 GPCRs are the largest family of cell-surface receptors
o They play a role is a wide variety of functions, including but not limited
to: embryonic development, neurotransmission and sensory
perception
 They consist of :
o 7 alpha-helical membrane spanning domains
o Extracellular domains which form the ligand binding site
o Intracellular domains which interact with G proteins
 G proteins can be:
o heterotrimeric - composed of three different protein subunits: an alpha subunit, a beta
subunit, and a gamma subunit
o or monomeric – composed of a single subunit most similar to the alpha subunit above
 A G protein acts as an on/off switch or timer:
o If GDP is bound to G protein, it is inactive
o G protein is active when bound to GTP
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How G Proteins Work
1. Binding of a ligand to the GPCR changes its shape allowing it to associate with a G protein
2. The G protein is then activated by the exchange of GDP for GTP
3. The GTP-bound alpha subunit and the beta-gamma dimer dissociated from the GPCR
o They can each relay messages in the cell by interacting with other membrane bound
proteins involved in signal transduction
4. The GTPase activity of the alpha subunit, hydrolyzes GTP returning it to its inactive GDP
bound state, ready for the next cell signalling event

Key Subtypes of G Proteins and their Effectors. Diversity

of

GPCR Responses

Targeting GPCR Function

 Between 30 and 60% of medications used today target GPCRs
 Many bacteria secrete toxins which make us ill by interrupting specific G protein-linked
receptor functions
o Example - Cholera toxin enters intestinal cells, where it modifies a G protein that controls
the opening of a chloride channel causing it to remain continuously active. This results in
large losses of fluids from the body and potentially fatal dehydration as a result

Enzyme-Linked Receptors
 They are cell-surface receptors with intracellular domains that are associated with an
enzyme, either directly or indirectly
o When a ligand binds to the extracellular domain, a signal is transferred through the
membrane,
activating
the enzyme
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Receptor Tyrosine Kinases (RTKs)

 RTKs are one of the main types of enzyme-linked receptors
o A kinase is an enzyme that catalyzes the transfer of phosphate groups
o RTKs transfer phosphates to tyrosine residues in proteins
 A single RTK can activate multiple signal transduction pathways and therefore cellular
responses
 Abnormal function of RTKs is associated with many cancers

How RTKs Work

 Two adjacent, inactive RTKs are depicted with a ligand approaching them
 Once bound to the ligand, the RTKs change conformation allowing them to dimerize and
activate the kinase activity
 The activated RTKs phosphorylate each other on their respective tyrosine residues
(cross-phosphorylation)
 Various intracellular proteins docked at each of the phosphorylated sites on the
RTK and participate in transduction of the signal within the cell
 The signal is terminated by a phosphatase that removes the phosphates from the
RTKs

Ligand-Gated Ion Channels

 When a signalling molecule binds to a ligand-gated
ion channel receptor, it changes conformation, and
the gate opens allowing specific ions through its
channel
o These ions affect cellular activity eliciting a cellular response
o When the ligand dissociates, the channel once again closes They are particularly
important for
signalling amongst
Intracellular Signalling -> Signal Transduction
electrically excitable
 Signal transduction usually involves multiple steps.
cells.
o Multistep pathways are beneficial as they can amplify a signal and provides
more opportunities for coordination and
regulation of the cellular response
 There are 2 common ways to propagate a signal
within the cell:
o Protein phosphorylation and dephosphorylation
o Small molecules and ions second messengers

Protein Phosphorylation/Dephosphorylation
 In many pathways, the signal is transmitted by a cascade
of protein phosphorylation events
o Protein kinases transfer phosphates from ATP to
a target protein = phosphorylation
o Protein phosphatases later remove phosphates
from these proteins = dephosphorylation
 Phosphorylation and dephosphorylation acts as a molecular
switch, turning activities on and off
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Example – MAPK Cascade
 One of the most common intracellular signaling pathways triggered by RTKs is known
as the mitogen-activated protein (MAP) kinase cascade
o First the monomeric G protein, Ras, is activated
o Ras activates the first of 3 three serine-threonine kinases, which go on to
activate each other in sequence
o Ultimately MAPK phosphorylates many target proteins which produce the final
cellular response
 Many growth factors, including nerve growth factor and platelet-derived growth
factor communicate using this pathway

Regulation of Monomeric G Proteins

 GEFs - guanine nucleotide exchange factors facilitate GDP dissociation
o Allows it to quickly exchange GDP for a new GTP and reactivate
 GAPs – GTPase activating proteins stimulate GTP hydrolysis
o Shorten the time it stays active
 GDIs - guanine dissociation inhibitors prevent the release of GDP
o Keeps it inactive

 This (figure on the preceding slide) is the

core of the RTK-MAPK signaling pathway. All
signaling molecules in the core MAPK
signaling pathway are positive regulators of
their downstream targets. Each component
of the pathway sequentially activates the
next, as follows:

1. Signaling is typically initiated when

ligand binds to the extracellular ligand-binding domain of two RTK sub-units,
bringing them together so that the RTKs are close enough to phosphorylate each
other on tyrosine residues.
2. Grb2 is an adaptor protein that binds both to phosphotyrosines on the RTK and to
Sos, a guanine nucleotide exchange factor (GEF).
3. Once Sos is brought to the membrane, it causes the small G protein Ras to
dissociate from GDP and bind to GTP, thereby activating Ras.
4. G proteins, such as Ras, are described as a molecular switches because they
are self-inactivating. G proteins contain a GTPase domain that will eventually
hydrolyze the bound GTP to GDP, deactivating themselves.
5. GTP-bound Ras will bind to MAPKKK, activating it. Activated MAPKKK
phosphorylates MAPKK.
6. Phosphorylation of MAPKK activates it, causing it to phosphorylate MAPK.
7. A significant proportion of phosphorylated, activated MAPK enters the nucleus,
where it phosphorylates transcription factors, modulating gene expression.
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* The activation of the three kinases, MAPKKK, MAPKK and MAPK is an example of a
phosphorylation cascade.

Second Messengers
 The ligand that binds the initial receptor is a pathway’s “first
messenger”
 Second messengers are small, nonprotein, water-soluble
molecules or ions, that spread throughout a cell by diffusion
o Second messengers participate in pathways initiated by
GPCRs, RTKs and Ion channels
 Common second messengers include:
o Cyclic Adenosine Monophosphate (cAMP)
o Ions (e.x. Ca2+)
o Inositol 1,4,5 - trisphosphate (IP3)
o Diacylgylcerol (DAG)
 cAMP is one of the most widely used second messengers
o Many signalling molecules trigger its formation by activating adenylyl cyclase
(AC)
o an enzyme in the plasma membrane, which converts ATP to cAMP
o cAMP usually activates protein kinase A (PKA), which phosphorylates various
other proteins
o Termination of the signal occurs when the enzyme, phosphodiesterase,
converts cAMP into AMP

Calcium
 Ca2+ is another important second messenger
o Cells actively regulate Ca2+ concentration
o Cytosolic concentrations of Ca2+ are normally much
lower than the concentration outside the cell and
internal compartments such as the ER lumen
o A small change in the number of calcium ions within the
cytoplasm represents a relatively large concentration
change which triggers cellular responses
 Ca2+ release is often brought about by activation of the
inositol phospholipid signaling pathway
o Cleavage of a specific phospholipid
(PIP2) in the plasma membrane by the
enzyme phospholipase C (PLC)
produces two important second
messengers:
o inositol 1,4,5-trisphosphate (IP3)
o diacylglycerol (DAG)
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Types of Cellular Responses

 Ultimately, a signal transduction pathway leads to regulation of one or more cellular
activities
 Responses may occur in the cytoplasm or in nucleus

Nuclear Responses
 Many signalling pathways regulate protein synthesis by turning genes on or off
o In such signaling pathways, the final molecule which is activated is a
transcription factor (TF)
o A TF is a protein which can bind to a specific DNA sequence regulating the
production of mRNA and ultimately protein
Cytoplasmic Responses
 Other pathways regulate the activity of proteins, rather than their synthesis
 For example:
o a signal could cause the opening or closing of an ion channel
o e.g. the inositol phospholipid signaling pathway
o Epinephrine alters glucose metabolism by activating glycogen phosphorylase,
which increases glucose levels
by stimulating glycogen breakdown

Regulation of the Response

 A response to a signal might not necessarily be simply “on” or “off”.

 There are several aspects of regulating, or fine-tuning, a cellular response to

consider
o Amplification of the signal (and thus the response)
o Specificity of the response
o Efficiency of the response
o Termination of the response

Signal Amplification
 Enzyme cascades amplify the cell’s response
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o At each step, the number of activated products is much greater than in the preceding
step
o e.x. - epinephrine signalling pathway
o - light-induced visual transduction cascade in rod cells

Specificity and Coordination

 Different cells produce different collections of
proteins
o These different proteins allow cells to
detect and respond to different signals
o Also means that the same signal can lead
to different responses in different cell
types

 Signaling cascades within a cell can

interact to affect multiple molecules in the
cell, leading to multiple cellular responses (e.x.
- secretion of substances from the cell, ion
channel opening, and transcription).

Signalling Efficiency
 Scaffolding proteins are large proteins to which other proteins are attached
o Cells increase signal transduction efficiency by grouping together the various proteins
involved in the same pathway using scaffolding proteins

Termination of the Signal

 Inactivation mechanisms are an essential aspect of cell signaling
 no signaling should go on forever!

TOPIC
6 – THE CELL CYCLE AND CANCER
Nucleic Acids
 There are two types of nucleic acids in the cell
o Deoxyribonucleic acid (DNA)
o Ribonucleic acid (RNA)
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 Nucleic acids are polymers also known as polynucleotides
 Each polynucleotide is made of monomers called nucleotides
 Nucleotides are the building blocks of nucleic acids
 Nucleotide Components – Sugar
o In DNA sugar is deoxyribose
o In RNA the sugar is ribose
 Nucleotide Components - Nitrogenous Bases
o There are two types of nitrogenous bases:
o Pyrimidines have a single six-membered ring
 cytosine, thymine, uracil A:T, C:G
o Purines have a six-membered ring fused to a five-
membered ring
 adenine, guanine

Structure of DNA
 Nucleotide monomers are joined by phosphodiester bonds to
form a polynucleotide
o The sequence of bases in a DNA or RNA polymer is unique for
each gene and always written 5’ to 3’
 DNA consists of two complementary polynucleotide strands
 The sugar phosphate backbones of the two strands are oriented antiparallel
(opposite) to one another (5’ -> 3’ and 3’ -> 5’)

Polynucleotide structure
 The structure of a polynucleotide is dictated by base pairing
 Hydrogen bonding allows compatible nitrogenous bases to
pair with one another
 DNA A=T and C=G
 RNA A=U and C=G

Organization of DNA
 A gene is a nucleotide sequence which codes for the production of a protein or functional
RNA molecule

Genes are found on Chromosomes

 Prokaryotes
o Genes are located on a single circular chromosome
o This DNA is associated with proteins responsible for packaging and condensing it into a
bacterial chromosome
 Eukaryotes
o Genes are spread across a series of linear chromosomes

DNA Packaging
 Eukaryotic DNA must be condensed to fit into the nucleus
 Its association with various proteins allows this to happen
o this DNA-protein complex is known as chromatin

Eukaryotic Chromosomes – Basic Structure

 In preparation for cell division, DNA is replicated, and chromosomes
condense
 Each duplicated chromosome consists of two sister chromatids (joined copies
of the original chromosome) held together by cohesin proteins
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 The centromere is the constriction that can be seen in the duplicated
chromosome, where sister chromatids are most closely attached
 During cell division, sister chromatids separate and move into separate
nuclei
 Once they have been separated, the sister chromatids are called
chromosomes

 Genome refers to the complete set of genetic information contained in an

organism's DNA.
 Karyotype – visualization of the full set of chromosomes belonging to an
organism
o The number of chromosomes vary widely between species
o This is not a reflection of complexity of the organism or even gene
number
o In humans:
o somatic cells (non-reproductive cells) have two sets of
chromosomes (2 × 23 = 46 total)
o gametes (reproductive cells: sperm and eggs) have half as many
chromosomes as somatic cells (23)

Cell Division
 Goal
o Pass identical genetic information to each daughter cell generated.
 Purpose
o Single-celled organisms use cell division to reproduce
o Multicellular organisms also use cell division for growth, maintenance and repair of cells
and tissues
The Cell Cycle
 Cell cycle refers to the life of a cell, from the time it is first formed by division of its parent
cell, until its own division into two daughter cells
Parts of the Cell Cycle
 Interphase accounts for ~90% of the cell cycle and can be subdivided into:
o G1 phase (“first gap”)
o S phase (“synthesis”)
o G2 phase (“second gap”)
 Cells grow during all three subphases of interphase, but chromosomes are duplicated only
during S phase
 Mitosis is conventionally divided into five phases:
o Prophase
o Prometaphase
o Metaphase
o Anaphase
o Telophase
 Cytokinesis overlaps the latter stages of mitosis.

 In addition to progressing through the cell cycle, cells may enter a nondividing state
known as G0
 Some cells enter G0:
o permanently
o e.x. - terminally differentiated neurons and skeletal muscle cells
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o for extended periods of time but can re-enter the cell cycle with appropriate stimulation
o e.x. - most liver cells
o and exit G0 repeatedly
o e.x. – lymphocytes and fibroblasts
 Cell cycle length varies enormously depending on the developmental stage and type of cell
o Almost all variability is found in the length of G 1 and G0

Regulation of the Cell Cycle

 The sequential events of the cell cycle are directed by a distinct cell
cycle control system which is analogous to a clock
 The “clock” has specific checkpoints at which the cell cycle pauses until
a go-ahead signal is received

Cell Cycle Checkpoints

 M-Checkpoint: Are Any Sister Chromatids Unattached? Checklist
summary:
o ✓ All sister chromatids attached to mitotic spindle
 G1-Checkpoint: Rest or Divide? Checklist summary:
o ✓ No DNA damage
o ✓ Sufficient resources
 S-Checkpoint: DNA OK? Checklist summary:
o ✓ No errors during DNA replication
 G2-Checkpoint: Fully Equipped? Checklist summary
o ✓ DNA without damage
o ✓ Chromosome set complete
o ✓ Enough cell components

G1 Checkpoint – are conditions favorable for cell division?

 Is there any DNA damage?
 Are there sufficient resources to see the cell through a round of division?
 If cells are allowed to proceed into S phase, they will almost certainly make it through the
other checkpoints
 If not, they can:
o Stop the cycle and try to fix the problem
o Enter G0 and wait for signs that conditions are more favorable
o Initiate cell death (apoptosis)
Regulation of the Cell Cycle
 The cell cycle control system is regulated by both internal and
external controls
 Internal regulators are proteins expressed by the cell itself which
act as negative or positive regulators at each checkpoint.
Positive Regulators of the Cell Cycle
 Positive regulatory proteins Cyclins and Cyclin dependent kinases
(Cdks)
 Different combinations of cyclins and Cdks regulate each checkpoint
 Cdks phosphorylate target proteins which carry out processes
needed to advance through the cell cycle
o They can only do this when associated with the appropriate
cyclin
 The concentration of Cdks remains fairly constant across the cell
cycle
 Where as the concentration of cyclins fluctuate across the cell cycle
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How Cdk activity is regulated

1. Cyclin concentration accumulates
2. Cyclin associates with Cdk
3. Cdk itself is phosphorylated, activating it
4. Cdk in turn phosphorylates target proteins
5. Target proteins allow for cell cycle progression
6. Cyclin is degraded by cytoplasmic enzymes

Negative Regulators of the Cell Cycle

 The best understood negative regulators are retinoblastoma protein (Rb),
p53, and p21
o They all function at the G1 checkpoint
 In the presence of DNA damage, p53 will activate p21, which inhibits the
cyclin/Cdk complex preventing entry into S Phase
 Rb regulates transcription via its interaction with E2F, a transcription factor.
 Interaction between Rb and E2F is regulated by its phosphorylation status which can be
modified by the G1/S cyclin-Cdk complex to promote cell cycle progression.

Regulation of the Cell Cycle

 The cell cycle control system is regulated by both internal and external controls
 External controls include signalling molecules such as growth factors/mitogens and other
survival factors.
 Example: Release of growth-promoting hormones, such as human growth hormone (hGH)
promote cell cycle progression
o A lack of hGH can inhibit cell division, resulting in dwarfism
o Too much hGH can result in gigantism

PDGF, Cell Signalling and the Cell Cycle: required for cell division in fibroblasts

Regulation of the Cell Cycle

 External controls also include growth conditions.
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 Cells require contact with the dish (or ECM in vivo) to trigger
division = anchorage dependence
 As an individual cell reaches its maximum surface area to
volume ratio, cell division will be activated
 When the density of cells becomes too high, cells will
withdraw from the cell cycle - contact or density-
dependent inhibition

Cancer and Loss of Cell Cycle Control

 Cancer often begins with a gene mutation that results in a faulty protein that regulates cell
reproduction
 Cancer cells do not respond normally to cell cycle control mechanisms (internal and/or
external) leading to either:
o too much cell division
o too little cell death

Oncogenes
 Positive regulators of the cell cycle are also known as proto-oncogenes
 Mutated versions of these genes ultimately INCREASE stimulation of the cell cycle and are
known as oncogenes
o Example - Ras

Tumor-suppressor genes
 Tumor-suppressor genes normally help prevent
uncontrolled cell growth
 Mutations that result in a decrease of tumor-suppressor gene
products may contribute to cancer onset
o Example – p53

Topic 7 – Replication

the HHMI chemical structure of DNA video

 The relationship between the structure and function of DNA became apparent once its
structure was determined
o Watson and Crick quickly predicted the semiconservative model of DNA replication
(Meselson and Sthal later confirmed it experimentally)
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o i.e. - during DNA replication, the parent molecule unwinds, each strand acts as a
template, and two new daughter strands are built based on

base-pairing rules

Replication
 3 key phases to consider: Nature Scitable – DNA replication
o Initiation
o Elongation
o Termination At the beginning of replication, DNA is unzipped by
helicase (yellow). Then primase (red) creates the first
few base pairs that DNA polymerase (blue) will
Replication - Initiation recognize, before it begins replication.

 Replication begins at sites called origins of replication, where two DNA strands are
separated, opening a replication “bubble”
 A bacterial chromosome will typically have one origin of replication
 A eukaryotic chromosome may have hundreds or thousands of origins of replication
 Replication proceeds in both directions from each origin, until the entire molecule is copied

 At the end of each replication bubble is a replication fork, a Y-

shaped region where new DNA strands are elongating

Helicases are enzymes that untwist the DNA double helix at the
replication forks.

Topoisomerase is a family of enzymes which correct the “overwinding”

ahead of replication forks by breaking, swiveling, and rejoining DNA
strands.

Single-strand binding proteins bind to and stabilize single-stranded

DNA in its linear form.
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Primase can start an RNA chain de novo and add RNA nucleotides one at a time using parental
DNA as a template.
 The resulting primer is short (5–10 nucleotides), and the 3 end serves as starting point for
the synthesis of DNA by DNA polymerase

Replication initiation summary

DNA Polymerase
 DNA synthesis is carried out by a family of
enzymes known as DNA polymerases
o They add nucleotides in the 5’-3’ direction

 DNA polymerase has 2 requirements:

 Free deoxynucleotide triphosphates (dGTP, dATP, dCTP and dTTP)
 A template strand of DNA with a short double stranded primer region
with an exposed 3’-OH

Replication - Elongation
 DNA polymerase catalyzes the formation of phosphodiester bonds, elongating the new
DNA strand
 As each new nucleotide is added to the growing DNA strand, it loses two phosphate groups as
a molecule of pyrophosphate
 The rate of elongation is about:
o 500 nucleotides per second in bacteria
o 50 nucleotides per second in human cells

Elongation
 The antiparallel structure of the double helix affects replication
 DNA polymerases add nucleotides only to the free 3end of a growing
strand
 Therefore, a new DNA strand can only elongate in the 5to3direction
 Along one template strand of DNA, DNA polymerase III synthesizes the
leading strand continuously, moving toward the replication fork

Synthesis of the Lagging Strand

 To elongate the other new strand, the lagging strand, DNA polymerase III
must work in the direction away from replication fork
 The lagging strand is synthesized discontinuously as series of segments
called Okazaki fragments
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 Synthesis of each Okazaki fragment begins with an RNA primer

generated by primase
 DNA polymerase III then completes the Okazaki fragment,
stopping and dissociating at the next RNA primer it encounters

 Upon completion of the next Okazaki fragment, the RNA

primers are removed and replaced with DNA by DNA
polymerase I
 DNA ligase seals the Okazaki fragments
generating a single continuous DNA strand

Synthesis of the Lagging Strand

Summary
Replication
Elongation
summary

Replication
Complex
 Protei
n s participating
in DNA replication form a large complex
known as the DNA replication machine or the replisome which significantly increases
the efficiency of the process.

Replication - Termination
 Limitations of DNA polymerase create problems for linear DNA of eukaryotic chromosomes
o The usual replication machinery provides no way to complete the 5 ends after the last
RNA primer is removed
o Therefore, repeated rounds of replication result in shortening of DNA molecules at the
ends
o Left unchecked this can lead to loss of genes from a chromosome
 This is not a problem for prokaryotes, as most have circular chromosomes
 Eukaryotic chromosomal DNA molecules have special nucleotide
sequences at their ends called telomeres
 Telomere shortening has been associated with aging

 Telomerase is a protein-RNA complex which recognizes the end of a

telomere sequence and elongates it (5’-3’) using its own built in RNA
template
 DNA polymerase then completes the other strand preventing
excessive shortening and loss of genes from the chromosome
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Topic 8Transcription and Translation

The Central Dogma of molecular biology …

 … is a framework for understanding the transfer of sequence information
between biopolymers (DNA, RNA and protein).

Transcription is DNA directed RNA synthesis

Translation is RNA directed

protein
synthesis

From an archive of Crick’s documents

Transcription – A Closer Look!
 The process of transcription is very similar to DNA synthesis
 Key differences:
o RNA polymerase carries out synthesis of the RNA
o The RNA transcript does not remained base paired to the template DNA
o Less accurate

There are many types of RNA. They are categorized based on their function:
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Transcription – Important Nomenclature
 The coding strand of a gene (5’-3’) is also referred to as the
non-template strand.
 Its sequence will be identical to that of the gene’s RNA transcript,
with T’s replaced by U’s
 The noncoding strand of a gene (3’ – 5’) is the template
strand for RNA synthesis.
 The RNA transcript is complementary to this strand

Transcription
 Genes can be
found on either
strand of DNA but
RNA synthesis
always occurs 5’
to 3’.

Transcription - General Steps

 Initiation
o RNA polymerase bind to the promoter region and opens the double helix to begin
transcription
 Elongation
o RNA polymerase adds complimentary nucleotides (A, U, C, G) to the 3’ end of the growing
RNA transcript
 Termination
o RNA polymerase and the RNA transcript dissociate from the DNA double helix

Prokaryotic Transcription

Initiation of Transcription
 Promotor contains regulatory sequences necessary for transcription initiation
 Transcription start site – location where transcription begins, denoted by +1
 Upstream – before the transcription start site, bases are given negative values
 Downstream – after the transcription start site, bases are given positive values

Initiation of Transcription - Prokaryotes

 Promoter - the DNA sequence found upstream of the transcription
start site that binds the transcriptional machinery
 There are 2 conserved elements found in prokaryotic promoters:
o -35 region
o -10 region
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Prokaryotic RNA Polymerase
 RNA polymerase is made up of multiple subunits.
 The σ (sigma) subunit recognizes the -35 and -10 regions
of the promoter of prokaryotic genes. This allows the
remaining subunits to associate with it and be properly
positioned to begin transcription at the +1 start site.
 The σ subunit dissociates from the polymerase after
transcription has been initiated.

Elongation Reaction
 RNA polymerase catalyzes the formation of
phosphodiester bond, adding NTPs onto the 3’ end of a growing
RNA molecule
 Unlike DNA synthesis, this process can begin without a primer

Prokaryotic Elongation
 During elongation, the prokaryotic RNA polymerase tracks
along the DNA template strand (3’-5’), synthesizing RNA in
the 5' to 3' direction
 RNA polymerase unwinds and rewinds the DNA double
helix as it progresses at a rate of ~ 40-50nt/sec

Prokaryotic Termination
 Prokaryotic genes have specific terminator
sequences
o There are two types of terminator signals:
o Rho-dependent (controlled by the Rho
protein)
o Rho-independent (controlled by sequences
in the DNA strand)
o xUltimately transcription of either one leads the RNA polymerase to disassociate from the
DNA and release the mRNA transcript

Transcription and Translation are Paired in Prokaryotes

 Multiple RNA polymerases can transcribe a single bacterial gene while numerous ribosomes
concurrently translate the mRNA transcripts into polypeptides
 In this way, a specific protein can rapidly reach a high concentration in the bacterial cell
Eukaryotic Transcription
 In eukaryotes different RNA polymerases make each type of RNA

In eukaryotes different RNA polymerases make each type of

RNA
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Initiation of
Transcription –
Eukaryotes
 Eukaryotic promoters have a conserved element known as a TATA box
 The TATA box allows for the association of several general transcription factors which
ultimately recruit and properly position RNA pol II
o The completed assembly of transcription factors and RNA polymerase II bound to a
promoter is called a transcription initiation complex

Eukaryotic Elongation
 RNA polymerase II synthesizes mRNA in the 5’ to 3’ direction
o *the elongation reaction itself is the same as was seen for prokaryotes
 Chromatin packaging presents a significant challenge with respect to the
movement of RNA polymerase
o The FACT (facilitates chromatin transcription) complex facilitates the
disassembly and reassembly of nucleosomes

Eukaryotic Termination
 At the end of each protein coding gene is a
polyadenylation signal sequence
 The RNA transcript is released 10 – 30 nucleotides past
this polyadenylation signal sequence via enzymatic
cleavage
o The RNA polymerase may continue on transcribing
hundreds or even thousands more basepairs before
it dissociates from the DNA template

Eukaryotic mRNA Processing

 Enzymes in the nucleus of eukaryotes modify the primary transcript in a process called RNA
processing before it is exported to cytoplasm
 During RNA processing, both ends of the primary transcript are usually altered
o The 5 end receives a modified nucleotide 5 cap
o The 3 end gets a poly-A tail
 The 5’ cap and poly-A tail share several functions:
o facilitate the export of mRNA to the cytoplasm
o protect mRNA from hydrolytic enzymes
o help ribosomes attach to the 5 end
Anatomy of a Eukaryotic Gene
 Eukaryotic genes are composed of both:
o Exons - Stretches of nucleotides referred to as coding regions because they are
eventually expressed and usually translated into amino acid sequences
o Introns - long noncoding stretches of nucleotides that lie between the coding regions
o Note - some introns contain sequences that may regulate gene expression

RNA Processing - RNA Splicing

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 Splicing removes introns and rejoins exons, creating an mRNA molecule with a continuous

coding sequence.
 RNA splicing is carried out by spliceosomes (ribozyme)
 Spliceosomes are made up of small nuclear ribonucleoproteins (snRNPs) that recognize
splice sites through sequence complementarity
and catalyze the splicing reaction

Alternative Splicing
 A gene can code for more than one polypeptide,
depending on which segments are treated as
exons during splicing
o This is called alternative RNA splicing
o ~95% of human genes undergo alternative
splicing

Prokaryotes vs Eukaryotes
Key Differences:
• In prokaryotes, translation of mRNA can begin before
transcription has finished
• In eukaryotic cells, the nuclear envelope separates transcription
from translation
• Eukaryotic RNA transcripts are modified through RNA processing
to yield the finished mRNA

Translation is RNA directed protein

synthesis

‘THE CODING
PROBLEM’
a.k.a – natures cryptogram

How can the linear sequence of information in nucleic

acids be converted to the linear sequence of polypeptides when they have such chemically
distinct subunits (nucleotides vs amino acids)?
TRANSLATION – A CHANGE IN LANGUAGE
3 nucleotides / aminoacid = 64
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Deciphering the Cryptogram
 Codon – a sequence of nucleotides which codes for an amino acid (or stop signal)
 Scientists had to contend with many questions:
o How many bases per codon? 3
o Was the code overlapping? nonoverlapping

The Genetic Code

 The set of rules specifying the correspondence between nucleotide triplets and amino acids in
proteins
o There are 64 codons:
o 61 code for amino acids, one of which is also a start
codon
o 3 are “stop” signals that end translation
 This code is used almost universally by all present day
organisms

Characteristics of the Genetic Code

 Unequivocal - each codon corresponds to only one amino acid
 Degenerate - for a given amino acid, there may be more than one codon

Reading Frame
 The reading frame, is the phase in which an mRNA sequence is read.
 There are always 3 possible reading frames but only 1 will yield the
correct protein!
 Start and stop codons are akin to punctuation for a sentence, determining
where translation begins and ends.

Mutations Can Affect Protein Structure and

Function
 Mutations are changes in the genetic code of an
organism
o Point mutations are changes in just one base pair of
a gene
o This may change an individual codon
o Frameshift mutation arise from insertions and
deletions of nucleotide pairs in a gene
o This alters the overall reading frame
 Mutations can lead to the production of an abnormal
protein
o If a mutation has an adverse effect on the phenotype of the organism, the condition is
referred to as a genetic disorder, or hereditary disease (e.g. – sickle cell disease)

Translation
 During translation the ribosome facilitates the matching of amino acid (AA)
charged transfer RNA (tRNAs) to the appropriate codon on the mRNA and
catalyzes the formation of peptide bods, joining the AAs into a polypeptide
chain.
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tRNAs Match Amino Acids to Nucleotide Codons
 A tRNA molecule consists of a single RNA strand that is about 80
nucleotides long
o Each carries a specific amino acid on one end
o Each has an anticodon on other end; the anticodon base-
pairs with a complementary codon on mRNA

tRNA Structure
 The flattened, secondary structure of a tRNA resembles a clover
leaf
 tRNA molecules actually twist and fold into a three-dimensional
conformation that is
roughly L-shaped

tRNAs and Wobble

base pairing
 Just like the genetic code, tRNAs have redundancies:
o The 20 AAs map to 61 different codons
o For some AAs there are more than one tRNAs/anticodons
o But some tRNA anticodons can base pair with more than one mRNA
codon
 Wobble base paring makes the latter possible
o wobble refers non-Watson and Crick base pairing
o This is tolerated in the 3rd position of a codon
o hence why so many codons for a given AA vary only in the 3 rd
position

 Before they can be used in the synthesis of a growing polypeptide

chain, tRNAs must linked with their designated AA
 This is carried out by the family of enzymes known as aminoacyl-
tRNA synthetases.
o Eukaryotes – 1 aminoacyl-tRNA synthetase / AA (therefore 20 in
total)
o This process requires energy!
Ribosomes
 Ribosomes facilitate specific coupling of tRNA anticodons with mRNA
codons
 The two ribosomal subunits (large and small) are made of proteins
and ribosomal RNA (rRNA)
o the ribosome is a ribozyme
 Bacterial and eukaryotic ribosomes are generally similar but also
have significant differences
o Hence why some antibiotic drugs specifically target bacterial
ribosomes without affecting eukaryotic ribosomes
 A ribosome has three binding sites for tRNA
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o P site - polypeptide
o A site - amino acid
o E site – exit
 The fundamental reaction carried out during translation is the formation of peptide bonds
o In each peptide bond, the carboxyl group of an AA is attached to the free amino group on

the incoming AA

Translation - Initiation
 Initiation brings together an mRNA, a tRNA with the first amino acid, and two ribosomal
subunits via the following steps:
o A small ribosomal subunit binds with a special initiator tRNA
o They associate with the 5’ cap on the mRNA and the small subunit moves along the mRNA
until it reaches a start codon (AUG)
o The large subunit then associates completing the translation initiation complex

Translation Elongation
 The addition of each AA to the C terminus of the
growing polypeptide occurs in three steps:
o codon recognition
o peptide bond formation
o translocation
 This process requires energy from GTP

Translation - Termination
 Termination occurs when a stop codon enter the A
site of the ribosome, leading to the following:
o The A site accepts a protein called a release
factor
o The release factor causes the addition of water
molecule instead of an amino acid
o This reaction releases the polypeptide, and the

ribosome dissociates
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Polyribosomes
 Multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome, or
polysome
 This enable cells to make many polypeptides very quickly!
Targeting Proteins to Specific Locations
 Polypeptide synthesis always begins in the cytosol
 Polypeptides destined for the ER or for secretion are marked by a signal peptide at their
amino-terminus
o A signal-recognition particle (SRP) binds to the signal peptide and brings it and its
ribosome to the ER

Protein Folding
 During and after synthesis, a polypeptide chain begins to spontaneously coil and fold into its
three-dimensional shape
 Once they have left the ribosomes, chaperone proteins may also assist a polypeptide to
reach its final mature 3D conformation

Post-Translational Modifications
 Some proteins undergo post-translational modification before
becoming fully mature/functional.
 These modifications may include:
o Enzymatic cleavage
o Aggregation/interaction of multiple polypeptides
o Addition of various moieties
o E.g. phosphorylation, acetylation, methylation, etc…

Topic 9 Regulation of Gene Expression

Regulation of Gene Expression

 …. refers generally to the set of mechanisms used by a cell to increase or
decrease the amount of a gene product it makes
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 Regulation may occur at any stage of gene expression
o i.e. – from the initiation of transcription through to degradation of a protein

Transcription Factors Regulate Transcription Initiation

 Transcription factors can be:
o Activators (positive regulators) which increases gene expression
o Repressors (negative regulator) which decrease gene expression
 Transcription factors recognize specific DNA sequences at or near the gene they regulate
Prokaryotes Organize their Genes into Operons
 An OPERON is a cluster of co-regulated genes
o The genes found in an operon are:
o functionally related
o under the control of a single promoter
o transcribed as a single mRNA but yield several separate functional molecules
 The name is derived from the cis-regulatory sequence which controls their expression – the
operator
 This is an efficient
mechanism by which
prokaryotes can
regulate a group of
related genes using
a single set of
signals to coordinate
their expression.

This mRNA is polycistronic – it codes

for more than one protein product

The Lac Operon

 The Lac operon consists of a set of genes required for the metabolism of lactose
 The Lac operon experiences both positive and negative regulation of transcription
 Its repressor LacI, is coded for by an independently regulated gene

Negative Regulation of the Lac Operon

 In the absence of lactose, LacI binds to the operator sequence, preventing expression of
the operon
 When lactose is present, it binds to the repressor, changing its conformation and allowing
for the expression of the operon’s genes
o You can say that the expression of the Lac operon is induced when lactose is present
 Removal of the LacI protein following lactose binding allows RNA polymerase to proceed
 The lac operon genes are then transcribed and translated
 The proteins produced are enzymes which ultimately breakdown lactose
 Following breakdown of lactose, the LacI can once again bind to the operator sequence
preventing transcription of the Lac operon genes

Genes Expression can be Regulated by Extracellular Signals

 In bacteria this often relates to molecules present in their growth medium, such as:
o Amino acids
o Carbohydrates
 Lactose isn’t the preferred fuel source of E.Coli, glucose is!
 Even if glucose and lactose are both present the cell will chose to continue to use glucose
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o But how?

Positive Regulation of the Lac Operon - Switching between

Glucose and Lactose
 cAMP levels relate to glucose availability
o when glucose is plentiful, [cAMP] = low
o when glucose is depleted, [cAMP] = high
 Catabolite activator protein (CAP) (also known as the cAMP receptor
protein CRP) binds cAMP
 The cAMP-CAP complex serves as an activator of the Lac operon
o Binding upstream of the promoter/operator for the Lac operon, it serves to enhance the
binding RNA polymerase and therefore its transcription

Activation of the Lac Operon


Regulation of the Lac Operon – Summary
Not exactly
true!
Some activity is
seen but the
dimmer switch
in turned to
“low”

Regulation of Gene Expression in Eukaryotes

 Most eukaryotic genes have multiple
control
elements beyond the promoter
 These are segments of noncoding DNA that
serve as transcription Dimmer
factor binding sites switch set to
 Control elements and “high”
the transcription
factors
they bind are critical to
the precise regulation
of
gene expression in
different cell types

 Proximal control
elements are located
close to the promoter
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 Distal control elements or enhancers, may be far
away from the gene or located in an intron

 To initiate transcription, eukaryotic RNA polymerase

requires the assistance of general transcription
factors
o as discussed in the transcription lecture
 High levels of transcription depend on control
elements interacting with specific transcription
factors
o An activator is a transcription that binds to an
enhancer and stimulates transcription
o Some transcription factors function as
repressors, inhibiting expression of a given
gene
 Distant regulatory proteins (both activators and repressors) are brought in proximity to the
RNA polymerase by DNA bending and form part of the transcription initiation complex via
interaction with mediator proteins

Coordination of Gene Expression in Eukaryotes

 Co-expressed eukaryotic genes are not organized in operons
o There are a few minor exceptions
 Co-expressed genes can be scattered over different
chromosomes, each with its own promoter and control
elements
 Activators recognize specific control elements and promote
simultaneous transcription of the genes
Combinatorial Control of
Gene Activation
 A particular combination of
control elements can activate
transcription only when the

appropriate activator proteins are present.

 This underlies differential gene expression in
different cells.

Manipulating Transcription Factors

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Regulation of Gene Expression
 …. refers generally to the set of mechanisms used by a cell to increase or decrease the
amount of a gene product it makes
 Regulation may occur at any stage of gene expression
o i.e. – from the initiation of transcription through to degradation of a protein
BIO 1140

OTHER NOTES

The cytoplasm of a cell possesses a different chemical composition than the extracellular environment. It is kept at a
different pH and possesses different proteins, molecules, and ions, that are involved in different chemical reactions
compared to the extracellular solution. For these cellular chemical reactions to occur properly, they must be maintained in
a very specific intracellular environment and therefore must be prevented from mixing with extracellular components. The
life of the cell depends on it. For this reason, a barrier is needed to separate intracellular contents from the extracellular
environment.

However cells must take in nutrients and expel waste so this barrier must also act as a selective gateway, allowing some
molecules to pass but not others. It must also be able to sense extracellular chemical signals. Moreover, cells must grow,
divide, and change shape, so this selective gateway barrier must also be able to change its surface area and be dynamic
and flexible enough to accommodate the wide range of shapes that cells exhibit.

This barrier, referred to as the plasma membrane, surrounds all cells in all forms of life. It is transparent under the light
microscope and is very thin, typically only about 5 nm thick. Compared to the size of the cell this is extremely thin. Think
of the plasma membrane as a thin sheet of hydrophobic molecules, specifically lipids, that prevents the mixing of the two
hydrophilic aqueous solutions on either side. The gatekeepers that regulate traffic across this lipid barrier are actually
proteins embedded in the lipid layer. Thus the plasma membrane is made of proteins embedded within a layer of
hydrophobic lipids.

Although the hydrophobic portion of the plasma membrane is impermeable to hydrophillic molecules including water, it is
important to note that hydrophobic molecules can still freely diffuse across.

Oil and water don’t like to mix because when two hydrophilic molecules come into contact, the forces between them are
attractive resulting in them spending more time in contact with each other. Whereas when a hydrophilic and hydrophobic
molecule come into contact, there are relatively little attractive forces between them and they therefore spend little time in
contact with each other.

To put this another way, hydrophilic molecules always prefer to be in a more energetically favorable configuration and
thus continually reposition themselves in order to attempt to always maximize their contact area with other hydrophilic
molecules. As a result, contact area with hydrophobic molecules is minimized. To an observer, this phenomenon appears
as if the hydrophilic molecules shun the hydrophobic molecules.

So how can such a large hydrophobic surface area exist around each cell if hydrophilic molecules are to be in the most
energetically favorable configuration? We will learn the simple answer to this in the next section.

in a previous section we learned about the selective hydrophobic barrier that separates intracellular contents from the
extracellular environment. However this hydrophobic layer is actually a bilayer created by the hydrophobic regions of two
adjacent lipid molecules. As such, the plasma membrane is said to be composed of a lipid bilayer.

Molecules such as these lipids that possess both hydrophilic and hydrophobic regions are called amphipathic.
Amphipathic molecules are very important for the formation of the lipid bilayer, as we will see shortly. Examples of
amphipathic molecules include phospholipids, glycolipids, triacylglycerol, phosphatidylethanolamine, and cholesterol.

So to answer the question from the previous section, how can such a large hydrophobic surface interface exist around
each cell if hydrophilic-hydrophobic interactions are to be kept to a minimum? The answer is quite simple: Amphipathic
molecules. Because amphipathic molecules possess both hydrophobic and hydrophilic regions, they essentially covalently
attach the hydrophobic-hydrophilic interface.

Imagine amphipathic molecules as hydrophobic molecules with a small hydrophilic head stapled to the top, which
essentially shields the hydrophobic region from the surrounding hydrophilic solution.

The hydrophilic molecules in the aqueous solution on either side of the lipid bilayer are just as happy interacting with the
hydrophilic heads of the amphipathic lipid molecules as they are with each other. You can see now how the size and
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shape of the lipid bilayer are free to change without affecting the energetically favorable interactions between the
hydrophilic molecules on either side

There are three main types of lipids found in the lipid bilayer of plasma membranes: Phospholipids, sphingolipids, and
cholesterol. The most abundant of these three groups are phospholipids. The name phospholipid is due to the phosphate
group that attaches a hydrophilic head group to its two hydrophobic lipid tails.

To be more precise, these two hydrophobic tails are attached to a glycerol molecule. The glycerol molecule is attached to
a phosphate group which in turn is attached to a hydrophilic head group such as choline or serine. The two hydrophobic
tails are each composed of a hydrocarbon chain, usually 15-22 carbons in length.

If every carbon in the chain is connected to its neighbor via a single bond, the tail would be straight. However the
presence of one or more double bonds creates bends, or kinks in the chain. Most phospholipids possess one straight tail
containing no double bonds and a second bent tail containing one or more double bonds. The tail with no double bonds is
said to be saturated because the carbon chain is attached to the maximum number of hydrogen atoms possible. A chain
with an absence of at least one hydrogen atom and corresponding double bond is said to be unsaturated.

Notice that the width of the phospholipid head is similar to the width of the tail region, resulting in a roughly cylindrical
shape. This cylindrical shape has an important implication for the side by side packing of phospholipids. However before
we examine this implication, let’s first have a look at a different type of amphipathic lipid: detergents.

Detergents are similar to phospholipids except that they possess only one hydrophobic tail instead of two. As such,
detergents have a shape resembling that of a cone rather than a cylinder. Multiple detergents in solution thus pack
together as cones which together create a circle, or in 3D, a sphere. Such a sphere is referred to as a micelle. Notice that
in a micelle all of the hydrophobic regions are concealed on the inside (with no empty space) while all hydrophilic regions
are on the outside, in contact with the surrounding hydrophilic solution.

Remember that the repositioning of these detergent molecules into a spherical shape is a result of the adjacent
hydrophilic molecules attempting to assume the most energetically favorable configuration. With all of the detergent tails
facing inward, hydrophilic-hydrophilic interactions are maximized.

If we substitute the detergent for a phospholipid we essentially change the shape of the molecule from a cone to a
cylinder. If we pack these cylindrical phospholipids side by side, we see that adjacent cylinders form a flat plane rather
than a sphere. In order to shield all of the hydrophobic regions from the surrounding hydrophilic solution, phospholipids
must also assemble on the opposite side, in the opposite orientation. This results in two planes that interact back to back,
with all hydrophobic regions blocked from interacting with any hydrophilic molecules.

The only exception to this would be the edges of the sheet. To solve this problem, the edges of the sheet can join with
each other to form a continuous sphere. The lipid bilayer thus forms a spherical barrier around the entire cell. In fact,
when phospholipids are added to water they spontaneously rearrange to form such spheres. These spheres are called
liposomes and can range from 25 nm to 1 mm in diameter. Notice that unlike micelles which are formed from a monolayer
of lipids, liposomes are formed from lipid bilayers.

Remember that it is the hydrophilic interactions that maintain the integrity of the hydrophobic barrier. There are benefits to
this feature of the lipid bilayer. For example, a tear in the plasma membrane that exposes hydrophobic edges would close
up very quickly.

All cells possess a plasma membrane. Simple bacteria possess a single membrane around the entire cell whereas
eukaryotes possess additional plasma membranes around the nucleus and other organelles. Examples of
intracellular compartments that possess plasma membranes include the nuclear envelope, endoplasmic
reticulum, Golgi, mitochondria, chloroplasts, and lysosomes. For these compartments, the plasma membrane
acts as a selective barrier just as it does for the entire cell. However the plasma membranes that surround these
compartments each have a different function, and this is typically represented by different collections of plasma
membrane proteins.

Regardless of their differences, all plasma membranes have at least one function in common: They must
compartmentalize the aqueous contents inside (via their hydrophobic lipid bilayer) and act as a selective,
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permeable barrier that allows some molecules to cross over but not others (via their plasma membrane proteins).
The plasma membrane proteins can also actively pump molecules into or out of the cell or compartment.

Other plasma membrane functions include responding to external chemical signals, interacting with adjacent
cells, and acting as a scaffold for biological activity. These functions are all carried out by plasma membrane
proteins.

The plasma membrane can be thought of as a two-dimensional fluid. Just as aqueous molecules diffuse in three
dimensions throughout a three dimensional fluid, plasma membrane lipids and proteins diffuse in two dimensions
throughout the lipid bilayer. Because of this, the plasma membrane is not uniform in composition. Different lipids and
different proteins are present throughout the membrane, giving the plasma membrane a mosaic appearance. The plasma
membrane can thus be thought of as a fluid mosaic.

In fact, individual phospholipids within the bilayer can rotate as fast as 20,000 rpm and diffuse laterally at a rate of 2
μm/sec. Yet despite the highly fluid nature of these lipids, they rarely switch from one side of the bilayer to the other. Such
“flip-flop” events are so infrequent that they occur less than once every 30 days for any individual lipid.

The fluidity of the plasma membrane is very important as it allows proteins to rapidly diffuse from one area to another. It
allows membranes to fuse, mixing their components together. It allows the membrane to grow when cells grow and to
change shape accordingly. New lipids can be added without breaking continuity. The composition and distribution of both
lipids and proteins can also constantly be regulated and can change rapidly based on the needs of the cell. This fluid
mosaic is therefore also highly dynamic.

Several factors affect the degree of fluidity within the lipid bilayer. Let’s examine some of the important factors.

1.Temperature
As the temperature increases, plasma membrane fluidity increases as well. As the temperature decreases, fluidity also
decreases.

2.Number of double bonds in phospholipid tails

In the absence of any double bonds, phospholipid tails are able to pack tight against each other. This limits their fluidity.
The presence of one or more double bonds results in more free space between the tails of adjacent phospholipids, thus
allowing them to move more freely. Therefore increasing the number of double bonds in phospholipid tails leads to
increased fluidity.

3.Phospholipidtail length
Similar to the effect of the number of double bonds on membrane fluidity, decreasing phospholipid tail length also results
in more space between adjacent tails, and therefore increases plasma membrane fluidity

4.Cholesterol
In animal cells, about 20% of the lipid bilayer is composed of the amphipathic lipid cholesterol. Cholesterol fills in the gaps
between the tails of adjacent phospholipids. Increasing the amount of cholesterol in the lipid bilayer therefore decreases
plasma membrane fluidity.

Because there are multiple factors influencing the fluidity of the plasma membrane, cells can adjust some of these factors
in order to maintain fluidity at a constant level. For example, most single-celled organisms must constantly adapt to
varying environmental temperatures. So if the extracellular temperature decreases, cells can compensate by incorporating
phospholipids with more double bonds into their plasma membranes in order to keep membrane fluidity constant.

Lipid rafts are regions of the plasma membrane that travel and diffuse through the bilayer as one unit. They possess
about four times as much cholesterol and contain more sphingolipids than the surrounding lipid bilayer. Double bonds are
still present in the phospholipid tails of lipid rafts but to a much lesser extent compared to the surrounding phospholipids.
Because of all of these factors, phospholipids in lipid rafts are much less fluid than the rest of the lipids in the surrounding
bilayer.

Also, because of the greater number of hydrogen-saturated carbon atoms in lipid rafts, these phospholipid tails are on
average more straight, and the phospholipids are thus, on average, taller than the surrounding phospholipids. Lipid rafts
therefore appear to be thicker than the rest of the plasma membrane.
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Lipid rafts may have an important function in cellular signaling by concentrating certain proteins and regulating their
interaction with other non-raft proteins. Having many proteins clustered together may be important for signal amplification
of certain signal transduction pathways.

However it is important to note that the existence of lipid rafts in vivo is still controversial, and that this is still an active
area of research.

The composition of lipids and proteins on either side of the plasma membrane is asymmetrical. In terms of the lipid
bilayer, different phospholipids are found in different amounts on one side versus the other. For example, the intracellular
side possesses more phosphatidylserine whereas the extracellular side possesses more phosphatidylcholine. Inositol
phospholipids, which are important for intracellular signaling events, are found only on the intracellular side.

The same is true for plasma membrane proteins. Some are present only on the intracellular side whereas others are
present only on the extracellular side. Even proteins that span through both lipid layers are always present in the same
orientation, with one side facing the extracellular environment while the other side faces the cytoplasm.

One important plasma membrane asymmetry worth noting is the presence of carbohydrate molecules on the extracellular
or intra-organellar side but not on the cytosolic side. Such carbohydrate molecules are usually found covalently attached
to proteins (referred to as glycoproteins) however about 10% are instead attached to lipids (referred to as glycolipids). In
animal cells, there are usually enough glycoproteins and glycolipids in the plasma membrane to create a continuous coat
of carbohydrates that surrounds the entire cell. We will learn more about these plasma membrane carbohydrates in a later
section.

Plasma membrane proteins associate with the lipid bilayer in one of two ways: Integrally or peripherally. Integral
membrane proteins are ones which are permanently associated with the lipid bilayer. This includes transmembrane
proteins that penetrate both layers of the lipid bilayer, monolayer-associated proteins that are embedded in just one of the
two layers, and lipid-linked proteins that are held in place via covalent attachments to bilayer phospholipids. Both integral
membrane proteins and monolayer-associated proteins must possess hydrophobic domains in order to associate with the
hydrophobic region of the lipid bilayer. Naturally, just like phospholipids, these proteins are also amphipathic.

Peripheral membrane proteins are proteins that are transiently associated with the plasma membrane, usually interacting
with integral membrane proteins however they may also interact with the phospholipids directly.

Disrupting protein-protein interactions would lead to the dissociation of peripheral membrane proteins from the plasma
membrane while not disturbing the localization of integral membrane proteins. If one wishes to remove integral membrane
proteins from the plasma membrane (for example, in order to study protein function), the membrane must be disrupted via
the addition of detergents. Detergent molecules insert into the lipid bilayer and because of their cone shape discussed in a
previous section, create bends in the otherwise flat plasma membrane. This eventually leads to the dissolving of the
plasma membrane into small pieces, freeing the integral membrane proteins.

The most common type of integral plasma membrane proteins are transmembrane proteins, which we will learn about in
the next section.

Transmembrane proteins possess three distinctive domains: A hydrophilic intracellular domain, a hydrophilic extracellular
domain, and a hydrophobic transmembrane domain. Because the intracellular environment is chemically different from the
extracellular environment, the two corresponding hydrophilic domains of transmembrane proteins are also usually
chemically different. This chemical difference is a result of the different amino acid residues present in either domain.
Moreover, these two domains may have different functions. For example, the extracellular domain may function as a
signal receptor while the intracellular domain may function as an enzyme.

For some proteins, the function of the transmembrane domain might be to couple together extracellular and intracellular
functions. For example, in order to link extracellular signal reception to intracellular enzymatic activity.

Peripheral membrane proteins usually associate with the plasma membrane via interactions with either the extracellular or
intracellular domains of transmembrane proteins.

It would feel as if the transmembrane protein were trying to remain within the lipid bilayer because the phospholipid heads
would be trying to fill up the vacant adjacent interactions by attempting to interact with the hydrophilic domain of the
protein.
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If you were to continue pulling with more force in order to overcome this resistance, the entire lipid bilayer would be
dragged up along with the protein. This should give you some idea of the strength of these hydrophilic interactions,
something that we don’t normally appreciate when such forces are absent.

Also remember that the plasma membrane is fluid, so you could conceivable move the transmembrane proteins side to
side, within the plane of the lipid bilayer, and continue to observe the resistance of the surrounding phospholipids pulling
on the transmembrane protein.

Although this is simply a thought experiment, the same concept applies for how transmembrane proteins work with other
intracellular and extracellular proteins in order to shape the plasma membrane.

Transmembrane proteins possess hydrophobic transmembrane domains that interact with the hydrophobic region of the
lipid bilayer. As explained in the previous section, the presence of this hydrophobic region holds the protein in place,
however the protein is of course free to diffuse along the plane of the lipid bilayer.

These transmembrane domains are usually composed of either a single alpha helix, multiple alpha helicies, or a beta
barrel, formed from multiple beta pleated sheets. Let’s examine each of these three situations in more detail.

Transmembrane alpha helicies possess hydrophobic residues around the exterior of the helix and hydrophilic residues
concealed on the inside. Naturally, the hydrophobic residues interact with the hydrophobic layer of the lipid bilayer. Alpha
helicies represent the simplest type of transmembrane domain.

Some transmembrane domains are composed of multiple alpha helical domains, either packed together to form a thick
stalk, or arranged in a circle to create an aqueous channel that spans the lipid bilayer. Can you predict which domains of
either the stalk or channel would be hydrophobic or hydrophilic?

Beta barrels form channels similar to those formed by alpha helicies, however there is one important difference: Channels
composed of alpha helicies can be formed from a varying number of alpha helicies resulting in channels with varying
widths. On the other hand, the beta sheets that line the beta barrel interact with each other at a specific angle, essentially
locking the width of the central channel to a fixed diameter.

The purpose of this lesson is to learn about the diversity of transmembrane proteins, both structurally and functionally.
Each transmembrane protein is a machine, and just like in our world, different machines have different structures in order
to carry out their respective functions.

Rhodopsin
Rhodopsin is found in our retinas. When it detects light, its seven transmembrane alpha helicies shift position relative to
each other and this conformational change triggers an intracellular signaling cascade. This signal travels along neurons to
the brain which ultimately allows us to see light.

Epidermal growth factor receptor (EGFR)

The binding of the extracellular signaling protein epidermal growth factor (EGF) to the EGF receptor (EGFR) leads to a
conformational change in the extracellular EGFR domain. This allows two, bound EGFR proteins to dimerize, leading to
activation of their intracellular kinase domains, resulting in the start of an intracellular signaling cascade that eventually
triggers the cell to grow. EGFR possesses a single transmembrane alpha helix.

Cadherin
In the presence of calcium, an extracellular cadherin domain from one cell binds to the extracellular cadherin domain of an
adjacent cell. Meanwhile, the intracellular domains attach to the actin cytoskeleton. In this way, cadherin proteins provide
a structural connection point between cytoskeletons from adjacent cells. Like EGFR, cadherins posses a single
transmembrane alpha helix.

ATP synthase
ATP synthase is a molecular turbine composed of more than 20 polypeptide chains. Because of an asymmetrical
distribution of protons across the mitochondrial inner membrane, these protons flow down their electrochemical gradient,
turning the transmembrane domain of ATP synthase as they flow. Conceptually, this is similar to a waterfall turning a
turbine. As the transmembrane domain turns, it rotates a rotor, which in turn changes the conformation of six head
domains sequentially. This energy, in the form of conformational changes, is used to create ATP from ADP and Pi.
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Smooth er – lipid production
ER = Making proteins these proteins are particular types typically the transmembrane proteins although not
always the rough endoplasmic reticulum is also capable of modifying proteins to high carbohydrate groups to
them and that gives us glycoproteins such as the one that we can see right here in that diagram the green
structure that you can see on the surface of the protein pointing towards the inside of the vescicle is the
carbohydrate we're going to keep an eye on those carbohydrates as we consider the movement of molecules
through this and no memory system so having synthesize lipids and proteins the endoplasmic reticulum
packages of indexicals and those vesicles fuse with the golgi apparatus specifically they fused with the CIS
surface of the golgi apparatus you can see those same proteins and lipids joining that golgi and the golgi is
capable of sorting and packaging them not really that key function that we learn about with the golgi the golgi
however also synthesizes carbohydrates and is capable of further modifying some of these components before
they leave the golgi this would include proteins so this protein that I'm drawing a box around here has a more
elaborate green structure on its internal surface so you can see it has had more carbohydrate groups added to it
the golgi is also responsible for glycosylated lipids and so we get glycolipids such as the one on the other side
of the golgi there once it has finished its sorting packaging John it puts those molecules into vesicles and those
vesicles leave from the TRANS surface of the golgi apparatus these vesicles can be tested for different locations
in the cell the one we're looking at right here is what we refer to as a secretary vesicle so it will ultimately fuse
with the plasma membrane and release its internal components into the extracellular space of felt or the space
around that so and so I want to now consider the individual lipid and protein components and what happens to
the following this Fusion so you can see here that the lids that were at one point surrounding that vesicle now
have fused with possible memory this is where they are joining right here and now becoming continuous with
them and this is how our memory grows you also know about endocytosis of the pinching off of vesicles toys
the inside of the cell surface can shrink or remove components from the plasma membrane the membrane
components that were part of the inner surface of our vesicle so in our image these are the ones that are denoted
in the teal colour on the inner leaflet said that colour effect it doesn't show up at all it's changed the ones right
here on the inner leaflet I leave them in purple on the diagram there till they become fused with the outer leaflet
of the puzzle number so you can see nto matches up with teal and that means that any of the lipids that were in
that leaflet are presented to the outside and again we know the lipid bilayer is asymmetrical we have certain
types of living inside and outside it also means that any proteins or glycolipids orlikow proteins are going to be
presented to the outside and so whenever you're looking in the ER or the golgi or the vesicles the carbohydrate
components there little brain structures were pointing to the inside of those vesicles we can now see them on the
cell surface pointing towards the outside of the cell and again that's one of those features of the asymmetrical
nature of the membrane that you should remember the glyco component is always pointing towards the outside
of the cell similarly we can take a look at the inner leaflet of that membrane so when we look at the inner leaflet
this is the one that is in the tan colour that's right here and we look at that one that was on the outside once
they've used their on the inside and so that's really important to keep in mind and be able to follow where the
teal colour lids versus the tan colours let's go one other thing that isn't shown to us in this diagram is the
possibility of having membrane proteins that are integral membrane proteins or perform memory proteins that
are presented only to 1 surface of this also rather than having transmembrane protein we could have one of
these memories that is only presented to the outside of the cell or one of these memory proteins it's only
presented to the inner surface of the sun and so if we follow them along their journey through the ER to the
golgi to the vescicles and Alta summary they would similarly be associated with the appropriate leaflet of the
lipid bilayer

TOPIC 5

All organisms must detect signals in their environment and respond accordingly, in order to avoid predators, mate, and
acquire nutrients. Single-celled organisms accomplish this by detecting extracellular chemicals, which for example, may
direct them towards a food source, or away from predators. The extracellular signal is detected, the information is
processed within the cell, and the cell then undergoes the appropriate response.
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Multicellular organisms such as humans also sense and respond to the environment. For example, if it’s hot, we sweat. If
we smell food, we salivate. And when we approach a red stoplight, we stop the car.

The responses of a single-celled organism and a multi-celled organism to environmental stimuli may appear completely
different, however the underlying concept is the same. The main difference is that in multicellular organisms, there is a
division of labor among the cells. Some cells are responsible for sensing, some for interpreting the information, and some
for responding.

This detecting, interpreting, and responding pattern at the multicellular level, also exists simultaneously at the level of
each cell. That is to say, each cell also undergoes signal detection from other cells, information processing, and response.
It is therefore the combined and integrated effect of this communication at the cellular level between all cells in the body,
that give rise to the multicellular response we think of as sweating, salivating, or muscle movement. When looked at in this
way, we see that each cell in a multicellular organism detects signals, interprets the information, and elicits a response, in
a similar way as a single-celled organism.

There is one important difference however. While the goal of a single-celled organism is for the cell to survive, the goal of
a multicellular organism is for the organism to survive. This means that cells in a multicellular organism are not working for
their own survival, but rather for the survival of the multicellular organism.

Cell communication affects almost every aspect of cell structure and function, including cell shape, movement,
metabolism, gene expression, growth, and division. Cell communication is responsible for the events of embryonic
development and cell differentiation. It allows plants to be able to respond to light and dark and to flower. In fact, in a
multicellular organism, cells that do not receive any signals from other cells will by default, undergo apoptosis:
programmed cell death

Information is transmitted between cells in the form of molecules. A cell will send out a signal molecule to a target cell,
and that target cell will receive the molecular message, and respond in the appropriate manner. As you might imagine,
there are a multitude of different signaling molecules used in the human body for different purposes. These include
proteins, peptides, amino acids and derivatives including glutamate, glycine, acetylcholine, dopamine, epinephrine, and
thyroid hormone, as well as nucleotides, steroids, fatty acid derivatives, eicosanoids, and even dissolved gasses such as
nitric oxide.

Each signaling molecule is recognized by a specific receptor on a specific target cell. The receptor on the target cell then
converts the extracellular signal into an intracellular signal. This intracellular signal initiates events inside the cell such as
activation or deactivation of certain proteins, which leads to a response. The particular response will depend on the initial
signaling molecule, the receptor, the intracellular events, and the cell type.

Examples of responses include a change in gene expression, activation of metabolic enzymes, rearrangement of the
cytoskeleton, increased cell motility, DNA synthesis, survival, growth, differentiation, or even programmed cell death. Most
cells both send and receive signals, and therefore act as both signaling and targeting cells for different signals.

Despite the overwhelming number of different signaling events that occur between cells in the human body, each falls into
one of five main categories. These categories are based on the mechanism by which the signal molecule travels, as well
as the distance between signal and target cell.

Endocrine:
A signal molecule from a small group of localized endocrine cells is secreted into the bloodstream. The molecule is
therefore broadcast throughout the entire organism, making contact with every cell in the body. These signal molecules
are called hormones. These are important messages that usually require the action of multiple types of target cells. This is
called endocrine signaling.

Paracrine:
A cell releases a signal molecule into its local environment, the extracellular matrix, or ECM. The molecule then diffuses
locally to nearby cells. Because these signal molecules don’t enter the bloodstream, they only travel as far as diffusion will
allow, remaining in the local neighbourhood, and targeting only nearby cells. This is called paracrine signaling.

Autocrine:
Sometimes, paracrine signaling will also affect the original cell that sent the signal. That is, the cell that sends out the
signal also possesses the specific receptor for this signal molecule. This is called autocrine signaling.
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Cell-Cell communication:
Cells that are in direct contact can communicate with each other not by means of secreting signal molecules, but via the
interactions between their membrane-interacting proteins.

Neuronalsignaling:
Neurons can deliver messages over long distances to specific target cells extremely quickly. These electrical signals
travel down the axon of the neuron. At the end of the axon, the electrical signal is converted into a chemical signal, and
the signal molecules, called neurotransmitters, are released directly onto the cell membrane of the target cell.

As you can see from these different modes of communication, these messages are either public, private, or somewhere in
between.

Cells in multicellular organisms are exposed to hundreds of different signaling molecules from other cells. Each cell must
determine which information to detect, and which to ignore. For example, hormones travel throughout the entire body, but
are only intended to stimulate certain cells. All of the other cells could ignore this message. Whether a signal molecule is
detected or ignored is determined by the set of surface receptors present on the target cell membrane. Each cell
possesses a limited set of receptors, in order to listen for the signals that only they are concerned with.

Different cells that have the same receptor for a given signal molecule may react differently to the same signal, depending
on the cell type. This is because different cell types possess different intracellular signaling molecules. Therefore, the
effect of a target cell is not determined only by the signaling molecule, but also by the cell type, receptor type, and the
downstream intracellular signaling molecules that it possesses. This would be equivalent to someone sending out a memo
through the office that reads, “Everyone please prepare for Monday’s meeting”. Although everyone would have received
the exact same memo, different employees may have different tasks in preparation for Monday’s meeting. Remember, in
this situation each employee is working for the good of the company, and the same is true for cells within a multicellular
organism. For example, the signal molecule acetylcholine will cause heart muscle cells to decrease the rate of
contractions, while causing salivary gland cells to secrete saliva. The process of converting the extracellular signal into an
intracellular signal is called signal transduction.

Each cell possesses many types of receptors, with many copies of each, and is thus sensitive to a variety of signals.
However, a combination of signaling molecules does not always generate an overall outcome that is equal to the sum of
the individual effects. This is because there is an interaction between the different intracellular relay systems. This means
that the presence of one signal can modify the response to another signal. We will learn more about crosstalk
communication between different intracellular signal transduction pathways in a later section.

All organisms must detect signals in their environment and respond accordingly, in order to avoid predators, mate, and
acquire nutrients. Cell communication affects almost every aspect of cell structure and function. However despite the
overwhelming number of different signaling events that occur between cells in the human body, each falls into one of five
main categories: Endocrine, paracrine, autocrine, cell-cell communication, and neuronal signaling.

Each cell possesses many types of receptors, with many copies of each, and is thus sensitive to a variety of signals. Cells
must therefore determine which signaling molecules to detect and which to ignore.

Receptors are proteins that detect the extracellular signal and initiate downstream, intracellular events within the cell.
These downstream events usually involve a cascade of signaling proteins, each activating the next sequentially,
eventually leading to the cellular effect. The effect can be, for example, growth, cell division, motility, or gene expression.
This process can be extremely fast, occurring in milliseconds, or very slow, as in the case of gene expression, which
typically occurs hours after the receptor has been activated.

Some signaling molecules such as steroids and nitric oxide, are small and hydrophobic enough to cross the cell
membrane and bind to intracellular receptors. However the vast majority are not, and therefore bind to receptors located
in the cell membrane. We will focus on these cell membrane receptors, and discuss the intracellular receptors in a later
section.

Cell membrane receptors usually possess an extracellular domain, an intracellular domain, and a transmembrane domain.
The extracellular domain binds the signal molecule. The intracellular domain typically undergoes a conformational change
upon ligand-binding, thereby initiating the intracellular signaling cascade. The transmembrane domain that connects the
extracellular and intracellular domains. In this series, extracellular domains are colored shades of green while intracellular
domains are colored shades of blue.
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Each receptor can activate many target proteins, and each target protein can also activate many of its own target proteins,
thus resulting in significant amplification at each step of the signaling cascade. In this way, just a few extracellular
signaling molecules can result in thousands of activated intracellular signaling proteins. Moreover, one signaling protein
could result in the activation of several different types of downstream effectors, each leading to a different effect.

So just as different cells may respond differently to the same extracellular signaling molecule, different protein effectors
within each cell may respond differently to the same upstream activator. To put this another way, there is task delegation
among different cells in multicellular organisms, and also task delegation one level lower, within each cell.

There are three main categories of cell membrane receptors: ion-channel-coupled receptors, G-protein-coupled receptors,
or GPCRs, and enzyme-coupled receptors. Each type can be used in different types of signaling events such as
endocrine, paracrine, and neuronal signaling.

A. Ion-channel-coupled receptors

Ion-channel-coupled receptors regulate the flow of ions across the cell membrane. Different receptors regulate different
ions. They are quite varied in shape, but are usually composed of multiple subunits that surround a central channel
through which the ions flow. They therefore all possess transmembrane domains composed of multiple alpha helices. The
opening and closing of the central channel is regulated by the binding of a specific signaling molecule.

Ion-channel-coupled receptors are used by specialized cells such as muscle cells and neurons. The flow of ions across
the channel is driven by an electrochemical gradient, and can change the membrane potential in as quick as a few
milliseconds. As such, ion channels can convert a chemical signal, the signaling molecule, into an electrical signal, the
change in membrane potential.

B. GPCRs

Unlike ion-channel-coupled receptors which are varied in shape, GPCRs are all structurally similar. They are composed of
a single polypeptide chain which possesses an extracellular ligand-binding domain, seven transmembrane alpha helices,
and an intracellular, heterotrimeric G-protein-binding domain. In response to an extracellular signaling molecule, GPCRs
activate heterotrimeric G-proteins, which then transmit the signal through a downstream signaling cascade. In some
instances, the signaling cascade resulting from GPCR activation could eventually lead to the activation of an ion channel.

C. Enzyme-coupled receptors

The most common type of enzyme-coupled receptors are receptor tyrosine kinases, or RTKs. These receptors are
composed of two polypeptide chains, each possessing an extracellular ligand-binding domain, a single transmembrane
alpha helix, and an intracellular tyrosine kinase domain. However, this kinase domain is initially inactive in each monomer.
Binding of a signaling molecule to each monomer causes a conformational change in the extracellular domain, allowing
the extracellular domains to dimerize. Naturally, this brings together the intracellular kinase domains, resulting in their
activation. We will learn more about how this activation occurs in a later section.

D. Comparison of different types of receptors

The structure of these receptors is directly related to their function. To summarize the structural differences between these
three groups: Ion-channel-coupled receptors are usually composed of multiple subunits, GPCRs are composed of a single
polypeptide chain, and RTKs are composed of two polypeptide chains that dimerize only upon ligand-binding. Ion
channels and GPCRs have multiple membrane passes whereas each RTK monomer possesses only one. Although all
receptors have an extracellular ligand-binding domain, only RTKs possess intracellular kinase domains.

Some of the intracellular signaling proteins activated by these receptors act as molecular switches, toggling on or off, in
an active or inactive state. There are two main types of such switches; those that use ATP, via phosphorylation or
dephosphorylation, and those that use GTP, via GTP-binding proteins.

Phosphorylation is the covalent attachment of a phosphate group from ATP to a specific residue on a target protein. This
reaction is performed by proteins called kinases. In this series, ATP and phosphate molecules will be colored yellow, and
kinases will be colored red. RTKs initially use the ATP switch, via their intracellular kinase domains. The process occurs
as follows: First, an ATP molecule enters the active site of the kinase. Next, the kinase removes the third phosphate from
the ATP molecule and covalently attaches it to the target protein. Finally, the remaining ADP molecule diffuses away.
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The covalent attachment of a phosphate group alters the conformation of the protein, thereby leading to either activation
or deactivation, depending on the target protein. Kinases can phosphorylate one or multiple residues on the target protein.
The phosphate can then be removed by proteins called phosphatases. The level of activity of the target protein is
determined by the net effect of its specific kinases and phosphatases.

Kinases and phosphatases recognize their specific substrates with great accuracy. These target proteins can be anything
including other kinases or enzymes, ion channels, structural proteins, or transcription factors. In fact, 50% of
transmembrane and cytoplasmic proteins are phosphorylated at one or more sites.

There are two types of kinases: serine/threonine kinases phosphorylate serine or threonine residues on target proteins,
and tyrosine kinases phosphorylate tyrosine residues on target proteins.

Whereas the phosphorylation/dephosphorylation system can be compared to an on/off switch, GTP-binding proteins, also
known as simply G-proteins, instead work as molecular timers. They are turned on, or, activated, by the non-covalent
binding of GTP. In this series, GTP molecules are colored red, and G-proteins are colored orange. GPCRs initially
activate this GTP timer, through activation of heterotrimeric G-proteins.

Initially, the G-protein is bound to GDP from the previous activation cycle. Upon binding to one of several G-protein-
activating proteins, the G-protein releases the GDP. This allows a GTP molecule to bind, which activates the G-protein,
allowing it to interact with downstream targets. The ability of G-proteins to hydrolyse GTP is weak, and as such, several
seconds pass before the GTP molecule is hydrolysed to GDP and Pi. During this time, the G-protein remains active, and
continues to target downstream proteins. After several seconds have passed, the G-protein finally hydrolyses GTP. The Pi
diffuses away, while the GDP remains bound in the active site. In order to re-activate, the GDP must be released, which
can only be triggered by binding to another G-protein-activating protein.

There are two types of G-proteins: monomeric and heterotrimeric. Monomeric G-proteins are composed of a single
polypeptide chain that binds GTP, and can be either cytosolic or tethered to the inner membrane. Heterotrimeric G-
proteins consist of three subunits: an alpha subunit, a beta subunit, and a gamma subunit, and are always tethered to the
inner membrane. The GTP-binding domain of the alpha subunit is structurally similar to monomeric G-proteins. We will
learn more about these G-proteins in later sections.

GPCRs initially activate this GTP timer, through activation of heterotrimeric G-proteins, while RTKs initially use the ATP
switch, via their intracellular kinase domains. However both types of switches, ATP and GTP, can be used in both
pathways in later downstream signaling events.

Receptors are proteins that detect the extracellular signal and initiate downstream, intracellular signaling events within the
cell. There are three main categories of cell membrane receptors: ion-channel-coupled receptors, G-protein-coupled
receptors, or GPCRs, and enzyme-coupled receptors.

Some of the intracellular signaling proteins activated by these receptors act as molecular switches, toggling on or off, in
an active or inactive state. There are two main types of such switches; those that use ATP, via phosphorylation or
dephosphorylation, and those that use GTP, via GTP-binding proteins.

Whereas the phosphorylation/dephosphorylation system can be compared to an on/off switch, GTP-binding proteins, also
known as simply G-proteins, instead work as molecular timers. They are turned on, or, activated, by the non-covalent
binding of GTP.

G-protein-coupled receptors, or GPCRs, are cell membrane receptors that activate heterotrimeric G-proteins in response
to ligand-binding. They represent the largest family of cell-surface receptors. They are evolutionary ancient, as even
bacteria possess structurally similar proteins, although they don’t bind to heterotrimeric G-proteins. There are thousands
of different types of GPCRs in organisms ranging from yeast, to flowering plants, to mammals. Mice possess more than
1000 devoted to smell alone. In fact, GPCRs represent the largest superfamily of proteins in animals. As such, these
receptors are responsible for an enormous variety of cellular processes in different cell types. Humans possess over 800
GPCR isoforms involved in a multitude of processes including sight, taste, smell, neuronal communication, cardiovascular
regulation, endocrine activity, and reproductive functions. In fact, about half of all known drugs work through GPCRs.

Despite the wide diversity in function, GPCRs are all structurally similar. These proteins consist of a single polypeptide
chain that passes through the membrane 7 times. GPCRs are therefore sometimes referred to as 7 transmembrane
receptors, or 7TMs. The N-terminal domain is located on the extracellular side, while the C-terminal domain is located on
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the intracellular side. There are also three extracellular loops which are involved with ligand-binding, and three
intracellular loops, the third of which is involved with binding of the heterotrimeric G-protein.

When no ligand is bound, the hydrophobic interactions between the 7 transmembrane alpha helices stabilize the protein in
its inactive state. When a ligand binds to the extracellular domains, the transmembrane alpha helices shift with respect to
each other, thereby rearranging the intracellular domains. This leads to an increased affinity for the heterotrimeric G-
protein, which then binds the GPCR.

Monomeric G-proteins are composed of a single polypeptide chain that binds GTP. Heterotrimeric G-proteins on the other
hand, possess a similar GTP-binding subunit, referred to as the alpha subunit, as well as two additional subunits, referred
to as the beta and gamma subunits. The alpha and gamma subunits possess a hydrophobic motif that inserts into the
cytoplasmic side of the cell membrane, and as such, heterotrimeric G-proteins remain tethered to the membrane.

Many isoforms exist, each with different targets and each responsible for different downstream functions. For example,
humans possess genes for 16 alpha, 5 beta, and 12 gamma isoforms. Although the different isoforms are responsible for
different functions, they are all structurally similar. Moreover, different isoforms can come together in different ways, giving
rise to many combinations of heterotrimeric G-proteins.

When an extracellular signaling molecule binds to and activates a GPCR, a heterotrimeric G-protein, in complex with
GDP, will bind to the third intracellular GPCR loop. In some instances, chaperone proteins may bind to the C-terminal of
the GPCR to enhance heterotrimeric G-protein binding. This interaction causes a conformational change in the alpha
subunit, which displaces the bound GDP. This allows a GTP molecule to bind, activating the protein. The protein then
leaves the GPCR, and the alpha subunit detaches from the beta and gamma subunits. This happens many times, for as
long as the extracellular signaling molecule remains bound to the GPCR, leading to signal amplification. Both the alpha
subunit, and the beta gamma subunits, will go on to activate target proteins, thereby initializing the intracellular signaling
cascade.

Because they are tethered to the membrane, activated heterotrimeric G-proteins will activate membrane-bound target
proteins. These target proteins are either ion channels or enzymes. In the case of activated ion channels, the ion channel
itself is the effector, and the influx of ions represents an immediate cellular response to the extracellular signaling
molecule.

Unlike for ion channels, the membrane-bound enzymes targeted by heterotrimeric G-proteins are not themselves the
effector. Instead, these enzymes initiate an intracellular signaling cascade, eventually leading to the downstream cellular
effect. For this reason, the response time of these pathways is not as immediate as for ion channels. The longer the G-
protein is bound to the target protein, the more amplification occurs. However after a few seconds, the bound GTP will
eventually be hydrolysed to GDP, leading to inactivation. The two most commonly used membrane-bound enzymes
targeted by heterotrimeric G-proteins are adenylate cyclase, or AC, and phospholipase C, or PLC.

Heterotrimeric G-proteins are categorized into four groups based on which of these membrane-bound enzymes are
targeted, and which isoform of the alpha subunit is used. Heterotrimeric G-proteins that activate AC are categorized as
Gs, where the s stands for stimulatory. Those that inhibit AC are categorized as Gi, where the i stands for inhibitory. Gq
isoforms activate PLC, and not much is known about the fourth category, G12/13.

Remember that different cell types express different isoforms, and the same extracellular signaling molecule can therefore
lead to the activation of different types of heterotrimeric G-proteins in different cells.

Moreover, different isoforms of AC and PLC are used by a variety of cells for a variety of downstream events. Despite the
wide variety of G-protein, AC, and PLC isoforms, the core events of these signaling pathways are conserved.

Adenylate cyclase, or AC, is an enzyme composed of a single polypeptide chain. It consists of 12 membrane-spanning
alpha helices, two intracellular catalytic domains, C1a and C2a, and several linkers connecting the intracellular catalytic
domains to the transmembrane domains. C1a and C2a come together to form the catalytic core of the enzyme. AC is
activated by the interaction with the GTP-bound, active, alpha subunit of the Gs, heterotrimeric G-protein. This interaction
causes the catalytic domain of AC to convert ATP to cyclic-AMP (cAMP).

If the original extracellular signaling molecule is thought of as the primary messenger, cAMP can be thought of as a
secondary messenger. Although signal amplification occurs at several steps, including activation of the GPCR,
heterotrimeric G-protein, and AC, these events are all localized to the cell membrane. When cAMP is produced, it can
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freely diffuse throughout the entire cell. In pathways involving AC, the cAMP produced floods the cytosol, quickly
spreading the signal to a multitude of downstream targets. As you may by now imagine, different cells produce different
cAMP target proteins. Thus cAMP will have different effects in different cells.

One of the main downstream targets of cAMP is protein kinase A, or PKA. PKA is composed of two regulatory subunits,
and two catalytic subunits. In its inactive state, the regulatory subunits are bound to the catalytic subunits, thereby
inhibiting their function. When cAMP binds to the regulatory subunits, it induces a dramatic conformational change, which
releases the catalytic subunits. These catalytic kinases then phosphorylate serines and threonines on downstream target
proteins.

The other most commonly used membrane-bound enzyme targeted by heterotrimeric G-proteins is phospholipase C beta,
or PLC-beta. PLC-beta possesses a PH domain, and as such, is tethered to the inner cell membrane via the interaction of
the PH domain with inositol phospholipids. Certain phospholipids play key roles in the PLC-beta pathway. It is thus
necessary to take a short aside, in order to learn about the chemical structure of these phospholipids.

All phospholipids are composed of a hydrophilic head, a phosphate neck, and lipid tails. Hence the name, phospho-lipid.
Most phospholipids possess a small head group composed of either serine or choline. These phospholipids are referred
to as phosphatidylserine and phosphatidylcholine respectively. However a small minority of phospholipids possess a
larger head group, composed of an inositol ring. Inositol is composed of a 6-carbon-ring with several alcohol groups
attached. These inositol-containing phospholipids are thus referred to as phosphatidylinositols.

Some phosphatidylinositols possess two phosphate groups attached to the carbon ring, at carbon positions 4 and 5.
These phosphatidylinositols are referred to as phosphatidylinositol 4,5-bisphosphate, where bisphosphate means two
phosphates. Phosphatidylinositol 4,5-bisphosphate is usually shortened to PIP2.

When activated by the GTP-bound alpha subunit, PLC-beta cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into two
pieces. The head and neck together possess an inositol ring with three phosphate groups attached to carbons 1, 4, and 5.
This piece is thus referred to as inositol 1,4,5-trisphosphate, where trisphosphate means three phosphates. Inositol 1,4,5-
trisphosphate is usually shortened to inositol trisphosphate, or IP3.

The second piece, which is the remainder of the original phosphatidyl 4,5-bisphosphate (PIP2), is referred to as
diacylglycerol (DAG), where diacyl describes the two lipid tails.

Inositol phospholipids are also used in RTK-mediated signaling, however we will discuss those events in the RTK section.
When activated by the GTP-bound alpha subunit, PLC-beta cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into two
pieces; IP3, and DAG. Take a moment to remember the structure of PIP2, IP3, and DAG from part 1.

IP3 is a small, hydrophilic molecule, while DAG is hydrophobic. Therefore, once cleaved, IP3 diffuses rapidly through the
cytosol. It interacts with the IP3 receptor, a large tetrameric ion channel, located in the smooth endoplasmic reticulum.
This interaction results in the opening of the channel, and the release of Ca2+ from the smooth ER. The smooth ER is
used as a storage facility for Ca2+ in a variety of cells, for a variety of processes. Because Ca2+ is a small molecule that
rapidly floods the cytosol upon release, Ca2+ can also be thought of as a second messenger, similar to the production of
cAMP by AC.

The next player in this pathway is protein kinase C, or PKC. PKC is a cytosolic protein composed of several domains:
C1A, C1B, C2, and catalytic. Although these domains are all connected by flexible linkers, they are wrapped together
tightly. The ATP-binding cleft of the catalytic domain is therefore concealed, and inactive. When sufficient Ca2+ ions are
present in the cytoplasm, these ions bind to the C2 domain of PKC. This allows PKC to interact with the cell membrane.
The Ca2+ ions act as an ionic bridge between the C2 domain, and membrane phospholipids. Specifically, phosphatidyl
serine and phosphatidyl 4,5-bisphosphate (PIP2).

This interaction triggers the insertion of both C1 domains, consecutively into the membrane. These C1 domains interact
with multiple DAG molecules. This in turn, results in the unwrapping of the flexible linker connecting the C2 domain to the
catalytic domain, and the catalytic domain is then free to phosphorylate serine or threonine residues on target proteins.
Note that both Ca2+ and DAG are required to activate PKC. Although PKC is the most well-studied, other cytosolic
proteins possessing C1 domains will also be recruited to the DAG molecules in the cell membrane.

In this section, we will review all of the information we have learned about GPCR pathways using a specific example. This
example pathway will also introduce new players into the backbone pathway that we have just learned.
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Glucose is used as an energy source by all cells in the body, and as such, its concentration in the bloodstream must be
regulated to within a narrow range. This range is monitored and controlled by cells in the pancreas. A decrease in glucose
levels could result in loss of consciousness or a coma. An increase could result in loss of fluids and electrolytes in the
urine, eventually leading to serious health problems. One of the ways in which blood glucose levels are regulated is by
storing excess glucose in long polymers, called glycogen, in liver cells. When blood glucose is low, glycogen can be
broken down into glucose monomers, and when blood glucose is high, glucose can be polymerized into glycogen.

Glycogen breakdown in response to low blood glucose levels is regulated by a GPCR-mediated pathway, which we will
now examine in detail. Glycogen synthesis in response to high levels is regulated by an RTK-mediated pathway, which we
will examine in an upcoming section.

In response to low blood glucose levels, the alpha cells of the pancreas secrete the hormone glucagon. Glucagon is a
small protein composed of only 29 amino acids. Glucagon travels through the bloodstream to the liver, where it binds to
the N-terminal domain and three extracellular loops of the glucagon GPCR. This binding leads to a shifting of the 7
transmembrane alpha helices in the GPCR, resulting in a conformational change in the C-terminal domain and three
intracellular loops. The Gs heterotrimeric G-protein, in complex with GDP, binds to the third intracellular loop, resulting in
the release of GDP in exchange for GTP. The heterotrimeric G-protein dissociates from the GPCR, and the alpha subunit
separates from the beta and gamma subunits.

The activated alpha subunit diffuses through the membrane and activates the catalytic domain of AC, composed of C1a
and C2a, which converts ATP to the second messenger cAMP. An incredible amount of cAMP is produced for each
extracellular glucagon protein that is bound to the GPCR because of signal amplification occurring at multiple steps.

cAMP quickly floods through the cytosol and binds to the two regulatory subunits of PKA. These regulatory subunits
undergo a dramatic conformational change, releasing the two catalytic subunits. These catalytic PKA subunits are
serine/threonine kinases.

They phosphorylate three important target proteins: glycogen synthase, phosphorylase kinase, and CREB proteins.

Phosphorylation of glycogen synthase inactivates the enzyme, preventing the polymerization of glycogen from glucose
monomers. Phosphorylation of phosphorylase kinase, on the other hand, activates the enzyme. Phosphorylase kinase
then phosphorylates glycogen phosphorylase. The now active glycogen phosphorylase begins breaking down glycogen
polymers into glucose-1-phosphate monomers. These monomers are converted to glucose, and then diffuse into the
bloodstream.

Notice that phosphorylation of one enzyme, glycogen synthase, results in deactivation, while phosphorylation of another
enzyme, glycogen phosphorylase, results in activation. Also notice the large size of phosphorylase kinase. Although the
exact molecular structure has not yet been determined, cryo-electron microscopy experiments have revealed its general
size and shape.

While the activation or deactivation of these enzymes results in almost immediate effects, the catalytic subunits of PKA
also enter the nucleus to phosphorylate transcription factors. The most notable, CREB, or CRE-binding, bind to cAMP
response elements, or CREs, in the genome, promoting transcription of several genes encoding enzymes involved in
gluconeogenesis. This is of course a much slower response than the breakdown of glycogen to glucose, because it
involves gene transcription. The binding of glucagon to the glucagon GPCR in liver cells thus results not only in the
breakdown of glycogen polymers into glucose monomers, but also promotes the production of enzymes required to
synthesize glucose.

Even if blood glucose levels are within the normal range, the body may sometimes require an energy boost during
physical activity or stressful situations. When this occurs, beta-adrenergic cells in the adrenal glands secrete the hormone
adrenaline, now called epinephrine. Epinephrine is a small molecule derived from the amino acid tyrosine. Epinephrine
travels through the bloodstream and binds to beta-adrenergic GPCRs in the membrane of muscle cells. The same
pathway is activated, resulting in the production of glucose. The muscle cell then uses this glucose for energy.

Glucagon and epinephrine are not structurally similar, but both bind GPCRs and have the same intracellular effect. Thus
two different stimuli binding to two different receptors from different cells can have the same effect. This epinephrine
pathway also stimulates breakdown of triacylglycerols into fatty acids, which is an immediate energy source. This pathway
is very rapid. In skeletal muscle cells, glycogen is broken down within seconds of epinephrine binding.
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G-protein-coupled receptors, or GPCRs, are cell membrane receptors that consist of a single polypeptide chain that
passes through the membrane 7 times. GPCRs activate heterotrimeric G-proteins in response to ligand-binding.

Heterotrimeric G-proteins are composed of a GTP-binding subunit referred to as the alpha subunit, as well as two
additional subunits referred to as the beta and gamma subunits. The two most commonly used membrane-bound
enzymes targeted by heterotrimeric G-proteins are adenylate cyclase (AC) and phospholipase C beta (PLC-beta).

AC converts ATP into the second messenger cyclic-AMP (cAMP). One of the main downstream targets of cAMP is protein
kinase A (PKA). The glucagon-mediated response to low blood sugar levels uses this pathway.

PLC-beta cleaves PIP2 into IP3, and DAG. IP3 diffuses rapidly through the cytosol leading to the release of Ca2+ from the
smooth ER. Ca2+ ions then bind to PKC, which allows PKC to interact with the cell membrane, thereby activating the
enzyme.

Enzyme-coupled receptors are membrane-spanning proteins that are responsible for a multitude of cellular responses
such as growth, proliferation, differentiation, migration, and survival. These pathways involve several signal transduction
steps which eventually lead to alterations in gene expression, and the response times are thus typically slow, on the order
of several hours. However some receptors mediate cytoskeletal changes, thereby altering the shape and movement of the
cell, and these response times are much faster.

There are two main classes of enzyme-coupled receptors: receptor tyrosine kinases (RTKs), which contain 60 members
and are membrane-bound, and cytoplasmic protein-tyrosine kinases, which contain 32 members and are located in the
cytosol. RTKs are activated by extracellular growth and differentiation factors. For example, epidermal growth factor
(EGF) activates the EGF receptor (EGFR). Cytoplasmic protein-tyrosine kinases are activated indirectly and are
responsible for a diverse number of cellular functions including immune response, cell adhesion, and cell migration. Both
the membrane-bound and cytosolic enzymes phosphorylate tyrosine residues on target proteins. In this section we will
focus on the largest group, RTKs. Notice that while all of the other kinases we have learned about until now, including
PKA and PKC, are serine/threonine kinases, while RTKs are tyrosine kinases.

RTKs are composed of two single polypeptide chains each containing an extracellular, ligand-binding domain, a single
transmembrane alpha helix, and an intracellular tyrosine kinase domain. Binding of an extracellular signaling molecule
leads to dimerization of the extracellular domains of the two monomers. Depending on the specific pathway involved, this
dimerization may be caused by either the ligand or the receptor.

For example, in the case of epidermal growth factor (EGF), each EGF receptor (EGFR) monomer binds to one EGF
ligand, and the two EGFR monomers are held together via binding sites on the receptors. For platelet-derived growth
factor (PDF), dimerization is mediated not by the PDF receptor (PDFR) monomers, but rather by the two PDF ligands. It is
important to note that there are some exceptions to these two methods. For example, the insulin receptor consists of two
monomers that are covalently attached to each other via disulfide bonds regardless of the presence of the ligand insulin.
In this example, binding of insulin to its receptor is thought to result in a change in the relative positions of the two
transmembrane domains. This in turn is thought to lead to activation of the intracellular kinase domains.

Unlike GPCRs or ion channels where extracellular and intracellular domains are connected by multiple membrane
passes, the extracellular and intracellular domains of RTK monomers are connected by only a single membrane-spanning
alpha helix. Regardless of how elaborate a conformational change may be in the extracellular domain as a result of
ligand-binding, it is extremely difficult for this structural information to be translated across a single alpha helix. In the case
of GPCRs, the seven transmembrane alpha helices can shift position relative to each other, thereby relaying structural
information from one side of the membrane to the other. RTKs circumvent this problem by instead using dimerization for
activation.

Dimerization of the extracellular domains naturally brings the intracellular tyrosine kinase domains into contact with each
other. Each tyrosine kinase domain possesses a substrate-binding site, which will house an ATP molecule during a
phosphorylation reaction. Initially, this site is partially blocked by a region of the kinase called the activation loop. However
despite the blockage, the kinases are still able to function, albeit with a much lower rate of activity. This basal rate of
activity is enough to be able to phosphorylate a specific tyrosine residue in the activation loop of the adjacent monomer.
Each monomer therefore phosphorylates the other.

This trans-autophosphorylation of the critical tyrosine residue in the activation loop results in a repositioning of the loop,
exposing the substrate-binding site. Once this site is exposed, the enzyme is fully active and able to phosphorylate target
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proteins at a normal, active rate. These activated kinases then phosphorylate specific tyrosine residues in the C-terminal
tail of the adjacent monomer. These phosphorylated residues will become recruitment sites for downstream target
proteins, as we will see in the next section.

At this point, the signaling events will diverge into two pathways. First, the two fully active tyrosine kinase domains will
phosphorylate downstream proteins. Second, the two C-terminal phosphorylated tails will act as recruitment sites for
additional downstream target proteins. In total, dozens of proteins will accumulate at this site, each transmitting a
message along a different route to different cellular destinations.

The downstream effects of RTK pathways are thus complex and highly coordinated, which is fitting for the type of complex
cellular effects that these receptors are involved in, such as cell growth, differentiation, and proliferation

Although the specific proteins that will be recruited to the phosphorylated C-terminal tails of the activated RTK dimers will
depend on the specific pathway and cell type, there are some well-studied conserved key players that are involved in
many pathways.

For example, proteins that possess an Src-homology 2 (SH2) domain or phosphotyrosine-binding (PTB) domain will
recognize and bind to the phosphorylated C-terminal tail residues.

SH2 domains bind to phosphorylated tyrosine residues within the motif: Tyr-X-Asn. PTB domains on the other hand, bind
to phosphorylated tyrosine residues within the motif: Asn-Pro-X-Tyr. Although SH2 domains are generally conserved, PTB
domains are not.

Although the term ‘recruited” is used often in the scientific literature, remember that it does not mean target proteins will
somehow detect the phosphorylated C-terminal tail, and then begin making their way to the site. All proteins in the cell are
moving in a random direction and any encounter with an interacting protein is by chance. The term ‘recruited’ means that
when a chance encounter occurs, the two proteins remain bound to each other.

There are four main types of SH2 or PTB domain-containing proteins: Adaptors, docking proteins, transcription factors,
and enzymes.

Adaptor proteins:
Adaptor proteins connect RTKs with other intracellular proteins. As such, they must possess an SH2 or PTB domain to
interact with the tyrosine-phosphorylated C-terminal tail of the RTK, as well as another protein-protein-interacting domain
to connect with the intracellular target. A common example of such a protein-protein interaction domain is the SH3
domain. There are over 300 SH3 domain-containing proteins in humans, all of which bind to proline-rich motifs. A classic
example of such a SH3-domain containing adaptor protein is Grb2. We will learn more about Grb2 in a later section.

Docking proteins:
Docking proteins can be thought of as a USB hub attached to your computer. The device plugs into one USB port and
contains inputs for multiple USB devices, thereby increasing the number of USB devices that can connect with your
computer. In the same way, docking proteins contain an SH2 or PTB domain that binds to the tyrosine-phosphorylated
tails of activated RTKs. They also possess several additional phosphorylation sites. Once phosphorylated by other
kinases, these additional phosphorylation sites allow more SH2 or PTB domain-containing proteins to be recruited to the
area. An example of a docking protein is insulin receptor substrate (IRS), which we will learn more about in a later section.

Transcription factors:
Some transcription factors such as signal transducer and activator of transcription (STAT) dimers are also activated by
RTKs. Each STAT monomer possesses an SH2 domain and a tyrosine phosphorylation site. Once both monomers are
phosphorylated, they dimerize via the phosphorylated tyrosine residue from one monomer and the SH2 domain from the
other. The activated STAT dimer then enters the nucleus to regulate gene transcription.

Signaling enzymes:
Signaling enzymes include protein kinases, protein phosphatases, lipid kinases, and phospholipases. For example, PLC-
gamma is used in many RTK pathways and works in much the same way as the PLC-beta protein that we had learned
about in the GPCR pathway, cleaving PIP2 into IP3 and DAG. All of these enzymes possess SH2 domains, and hence,
bind to the tyrosine-phosphorylated C-terminal tails of activated RTKs.
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The most common signaling enzyme used in almost all RTK pathways is Ras. Ras is a monomeric G-protein tethered to
the cytoplasmic side of the cell membrane via a lipid tail. Ras, like all monomeric G-proteins, shares a structural similarity
to the GTP-binding domain of the alpha subunit of heterotrimeric G-proteins. Naturally, the cycle of GTP-binding and
hydrolysis is the same as that discussed in the heterotrimeric G-protein section. Just as timers are used in many aspects
of our lives such as cooking, racing, traffic lights, and final exams, these molecular monomeric G-protein timers are also
used in many cellular processes including cell division, differentiation, gene expression, cytoskeletal organization, vesicle
trafficking, and nucleocytoplasmic transport.

These timers can be regulated by accessory proteins to either speed up or slow down the clock, or to lock it in either an
active or inactive state. There are three types of such regulatory proteins: G-protein-activating proteins (GAPs), guanine
nucleotide-exchange factors (GEFs), and guanine nucleotide-dissociation inhibitors (GDIs).

GAPs speed up GTP hydrolysis, thereby shortening the amount of time that the G-protein remains active. GEFs stimulate
the release of GDP, thereby allowing the G-protein to quickly acquire a new GTP molecule and re-activate. GDIs inhibit
the release of GDP, thereby locking the G-protein in an inactive state. GAPs, GEFs, and GDIs are themselves regulated
by additional proteins, rendering the situation very complex.

In this lesson we will review all of the information that we have learned about RTK-mediated signaling events by using an
example pathway. This pathway will also introduce new players into the backbone pathway that we have just learned.

Platelet-derived growth factor (PDF) is involved in embryonic development, cell proliferation, migration, and blood vessel
formation. Epidermal growth factor (EGF) is involved in growth, proliferation, and differentiation. Both use the same core
pathway. We will use EGF as an example.

The binding of EGF to the EGF receptor (EGFR) leads to dimerization of the extracellular EGFR domains. This brings the
intracellular tyrosine kinase domains into contact with each other. The intracellular kinase domains phosphorylate each
other on the tyrosine residue of the activation loop, resulting in a conformational change that removes the loop from the
substrate-binding domain. With the active site revealed the enzymes are said to be fully active. Each kinase
phosphorylates specific tyrosine residues on the C-terminal tail of the adjacent monomer. The fully activated tyrosine
kinase domains and phosphorylated C-terminal tails then start a signaling cascade.

The adaptor protein Grb2 binds to a phosphorylated tyrosine residue on the C-terminal tail of the receptor, via its SH2
domain. The two SH3 domains located on the other side of Grb2 then recruit Son of sevenless (Sos) which is an activator
of Ras. Maintaining Sos in close proximity to the cell membrane allows Sos to activate Ras by triggering the exchange of
GDP for GTP.

The activated Ras protein then activates the kinase Raf. Raf phosphorylates the kinase MEK. MEK then phosphorylates
two kinases, ERK1 and ERK2. ERK1 and 2 then phosphorylate over 160 proteins in the cytosol and nucleus including
transcription factors, protein kinases, cytoskeletal proteins, apoptotic regulators, and other signaling proteins. This
eventually leads to the cellular effect of growth, proliferation, or differentiation.

In order to confuse undergraduates, each of these proteins is also sometimes referred to with a different name. Raf, MEK,
and ERK are also known as MAPKKK, MAPKK, and MAPK, where MAP stands for mitogen-activated protein. Humans
possess 14, 7, and 13 types of these kinases respectively. Just as different types of heterotrimeric G-proteins can be
formed by matching different combinations of subunits, different MAPK pathways can be created by matching different
combinations of MAPKKK, MAPKK, and MAPK proteins.

Another classic example of an RTK pathway is insulin. We had learned in a previous section about the glucagon GPCR-
mediated response to low blood glucose levels. Now we will examine the opposite pathway: The secretion of the peptide
hormone insulin in response to high blood glucose levels, which happens to be mediated by an RTK pathway.

High blood sugar levels results in the loss of fluids and electrolytes in the urine, eventually leading to serious health
problems. When blood glucose levels are too high, beta cells in the pancreas secrete insulin.

Each insulin receptor monomer is composed of two polypeptide chains: and alpha and a beta. These two chains are
derived from a single protein precursor via proteolysis. They are covalently attached to each other via disulfide bonds.
Unlike most RTKs, each monomer is also covalently attached to the other, also via disulfide bonds. This effectively locks
the receptor in a dimer configuration regardless of the presence of the insulin ligand.
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This receptor is also unlike other RTKs in that only one insulin ligand binds to the dimerized receptor.

The binding of insulin to the dimer results in a poorly understood conformational change that shifts the two
transmembrane domains with respect to each other. This causes the two intracellular tyrosine kinase domains to undergo
trans-autophosphorylation of specific tyrosine residues in the activation loop. Each activation loop contains three
phosphorylation sites and the resulting conformational change renders each kinase fully active. The kinases then
phosphorylate multiple tyrosine residues on the C-terminal tail of the opposite monomer, as well as tyrosine residues in
the juxtamembrane region.

Instead of directly recruiting proteins with SH2 domains, these tyrosine phosphorylation sites recruit insulin-receptor
substrates (IRSs). The IRS docking protein binds to phosphate groups in the juxtamembrane region via its PTB domain,
while its N-terminal PH domain most likely interacts with membrane phospholipids. The insulin receptor tyrosine kinase
domains then phosphorylate tyrosine residues in the C-terminal tail of IRS. The resulting phosphorylated residues on IRS
are then used to recruit SH2 or PTB domain-containing proteins. IRS effectively expands the number of phosphorylated
docking sites thereby allowing a greater number of SH2 or PTB domain-containing proteins to be recruited.

The SH2-domain-containing proteins PI3-kinase, Grb2, and Shp2 are recruited. Although recruitment of Grb2 and Shp2
lead to their own downstream effects including the recruitment of Sos and activation of Ras, in this section we will only
examine the PI3-kinase pathway since the Ras pathway was examined in the previous sections.

PI3-kinase has two subunits. One contains two SH2 domains and the other contains a catalytic kinase domain hence the
name PI3-kinase. PI3-kinase phosphorylates the third position on an inositol ring within inositol phospholipids. This
produces PI3,4-biphosphate (PIP2) and PI3,4,5-triphosphate (PIP3) which remain in the cell membrane. PIP2 and PIP3
provide docking sites for PH domain-containing proteins. The most notable of these PH domain-containing proteins are
phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB). It is important to point out that both PDK1 and
PKB are serine/threonine kinases, just like all of the other non-RTK kinases that we have learned about so far. Once
PDK1 and PKB are recruited to the membrane PDK1 phosphorylates PKB. PKB must then also be phosphorylated by
mTOR in order to become fully active.

The activation of PKB results in two main events. First, it leads to further downstream events which eventually result in the
translocation of GLUT4 channel-containing vesicles to the cell membrane. The insertion of GLUT4 channels into the cell
membrane allows glucose monomers to freely diffuse into the cytosol. Second, PKB phosphorylates glycogen synthase
kinase-3 (GSK-3) which deactivates this kinase. GSK-3 is a negative regulator of glycogen synthase (GS). Thus
unphosphorylated GSK-3 results in the inhibition of glycogen production from glucose monomers. Once phosphorylated
by PKB, GSK-3 releases its inhibition over GS and GS is thus allowed to synthesize glycogen.

RTKs are membrane-spanning receptors that are activated by extracellular growth and differentiation factors. Unlike PKA
and PKC which are serine/threonine kinases, RTKs are tyrosine kinases.

Binding of an extracellular signaling molecule to an RTK leads to dimerization, which is usually mediated by either the
receptor or the ligand. This leads to trans-autophosphorylation of the cytosolic kinase domain followed by phosphorylation
of the C-terminal tails. The phosphorylated C-terminal tails then act as recruitment sites for downstream SH2 or PTB
domain-containing proteins.

In the example that we have learned about, the phosphorylated C-terminal tails recruit Grb2, which then recruits Sos,
which then activates Ras. Ras activates Raf, which then phosphorylates MEK, which then phosphorylates ERK1 and
ERK2.

We have learned about GPCR and RTK-mediated signaling events as independent, isolated pathways, however in reality
each of these pathways is highly inter-connected with other pathways in the cell. This communication between different
signaling pathways is referred to as crosstalk. Crosstalk is important because the result of one pathway may depend on
other events occurring within the cell. For example, a signaling pathway that leads to cell migration may need to
communicate with another pathway involved in cell division such that when it is time to divide, cell migration is halted. A
good analogy would be the crosstalk that occurs between neural networks which allow the outcome of one event to be
influenced by other events within the brain. It is therefore important to think of intracellular signaling pathways not as linear
chains but as complex networks.
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Simple examples of crosstalk include diverging or converging signals. For example, the binding of EGF to the EGF
receptor activates the MAPK pathway which results in divergent downstream effects. On the other hand, the GPCR-
mediated CaMKII activation can converge with the RTK-mediated MAPK pathway via Ras activation by CaMKII.

We have learned about the RTK-mediated SH2-docking sites that lead to the activation of the Grb2, S-O-S, Ras, and
MAPK signaling cascade however these same events can also be activated by heterotrimeric G-proteins. We have also
learned about the mobilization of glucose in response to the intracellular second messenger cAMP however cAMP can
also inhibit growth in fibroblasts by blocking the MAPK signaling cascade. This occurs through the cAMP-mediated
activation of PKA which then inhibits Raf. The PKA and MAPK pathways can also both result in the phosphorylation of
CREB.

To complicate matters more, not only do different cells possess different downstream effectors, but there are usually
many isoforms of any given protein each perhaps leading to a different outcome.

In summary, the situation is much more complex than what we have discussed in these lessons.

DNA
DNA replication is conceptually simple. Because each of the two DNA strands are complementary to each other, each
single strand can serve as a template for the reproduction of the other. In other words, if we know the sequence of one
strand, we can determine the sequence of the other (Figure 1, Left). This is how DNA replication occurs.

You may remember that this same concept is used during transcription as RNA polymerase uses one DNA strand as a
template to produce a complementary mRNA transcript that matches the non-template strand (Figure 1, Right). However,
unlike transcription wherein only one new nucleotide strand is produced on one template, during DNA replication two new
nucleotide strands are produced at the same time, one on each of the two original DNA template strands.

This method of DNA replication is called semi-conservative because half of the original DNA is kept while the other half is
newly synthesized. The original, template strand is referred to as the parent strand while the newly synthesized strand is
referred to as the daughter strand. Because the two original DNA strands are anti-parallel to each other (with respect to
their 5′ and 3′ ends), DNA replication occurs in a bidirectional manner.

DNA replication occurs in a very similar manner in both prokaryotes and eukaryotes however in eukaryotes the process is
somewhat more complex. For simplicity, we’re therefore only going to focus on DNA replication in prokaryotes.

DNA replication begins with the binding of initiation proteins to DNA sequences called origins of replications (ORIs).
Because of their smaller genome, prokaryotes typically possess only a single ORI whereas eukaryotes possess multiple
ORIs on each chromosome (Figure 1).

The binding of these initiation proteins distorts the three-dimensional shape of the DNA in a way that pulls the two strands
apart. This results in the formation of a replication bubble and two replication forks, one at each end of the bubble (Figure
2, Left). The bubble shape is created by the two single-stranded DNA chains that have separated from each other and the
two forks are the transition points where the single-strands join back together into double-stranded DNA.

The opening of the replication bubble exposes the nitrogenous bases along the inside edge of the nucleotides in that
region (Figure 2, Right). This allows for the recruitment of two large protein complexes called replisomes which bind at
the two forks at either end of the replication bubble, in opposite orientations. The molecular machine within the replisome
complex that polymerizes the new DNA strand is called DNA polymerase. Each replisome contains two DNA polymerase
enzymes, one for each of the two DNA strands at that fork, as well as several accessory proteins that perform other,
supportive functions (Figure 3).

Once initiation is complete DNA replication is ready to begin. The two replisomes start to move away from each other in
opposite directions as they replicate the DNA (Figure 1, Left). Think of the movement of the replisome along the DNA as
the opening of a zipper. The replisome is the zipper handle that moves, the double-stranded DNA is the closed zipper
ahead, and the two single-stranded DNA chains are the open part of the zipper behind (Figure 1, Right).

The fork is the point at which double-stranded DNA separates into two single strands and the bubble, or in the case of the
zipper analogy, half bubble, is simply the shape formed by the two separated single strands of DNA. The fork is therefore
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not a fixed structure but rather a transition point that moves along with the replisome. Therefore just like a zipper, each
replisome separates double-stranded DNA into two single strands as it travels forward.

The molecular machine at the front of the replisome that separates the double-stranded DNA into two single strands is an
enzyme called helicase. Each of the two DNA polymerase enzymes then use one of these single strands as a template
upon which to synthesize a new, complimentary strand of DNA (Figure 2). DNA polymerase adds nucleotides at a rate of
four to several hundred nucleotides per second depending on the organism. It is extremely accurate, making less than
one mistake in a billion. When a mistake does occur, DNA polymerase has the ability to proofread and fix the mistake.

Each of the two original parent strands of DNA acts as a template for the polymerization of its new daughter strand. DNA
polymerase can only synthesize nucleotide chains in one direction, from 5’ to 3’, for the same reason as discussed for
RNA polymerase-mediated transcription (Figure 1, Left). That is, the chemical energy required to join the incoming
nucleotide to the growing chain is provided by the incoming nucleotide itself, within the bonds between the phosphate
groups at the 5’ end of the nucleotide (Figure 2). Because of this constraint, DNA polymerase must read the template
strand in the opposite, 3’ to 5’ direction.

This presents a problem for one of the two DNA polymerase enzymes. Because both enzymes are travelling in the same
direction, and because DNA is antiparallel, one enzyme would read one of the single-stranded DNA templates in the
correct 3’ to 5’ direction while the other would have to read the other single strand in the incorrect, 5’ to 3’ direction.

To mitigate this, the DNA polymerase on the incorrectly oriented strand moves away from the replication fork in the
correct 3’ to 5’ direction (Figure 1, Right). This means that if we were to follow the path of the correctly oriented strand
from the replication fork to its associated DNA polymerase, we would arrive at the front of the polymerase. However, if we
were to follow the corresponding path for the incorrectly oriented strand, we would arrive at the rear of its associated
polymerase. This setup ensures that both polymerases read their associated template in the correct 3’ to 5’ direction

The positioning and directionality of the two DNA polymerase enzymes and their associated single-stranded DNA
templates solves one orientation-associated problem but creates another. While one DNA polymerase travels towards the
replication fork as it travels down the correctly oriented strand (referred to as the leading strand), the other polymerase
travels away from the fork as it travels the correct way down the incorrectly oriented strand (referred to as the lagging
strand).

Because the lagging strand polymerase moves backwards relative to the replisome and replication fork, it doesn’t keep up
with the newly emerging lagging strand DNA that is continuously being generated by helicase. The lagging strand
polymerase is therefore forced to polymerize one short fragment at a time because it keeps having to go back to the start
of the next fragment each time it completes its current fragment. By the time it has finished one fragment, the next
fragment is ready to go, and the polymerase is therefore always behind, constantly trying to catch up.

Therefore, unlike the leading strand polymerase which never has to disassociate from the DNA as it continuously
polymerizes the new daughter strand, the lagging strand polymerase is forced to repeatedly associate and disassociate
from the DNA as it polymerizes its new daughter strand in short fragments. These fragments, named Okasaki fragments
after the biologist who discovered them, are a few hundred nucleotides long in eukaryotes but can be a few thousand
nucleotides long in prokaryotes. Because DNA polymerization occurs continuously on the leading strand but
discontinuously on the lagging strand, DNA replication is said to be semi-discontinuous (Figure 1, Left).

In reality, the lagging strand polymerase doesn’t really move away from the fork because the entire replisome moves as a
whole. Instead, the lagging strand DNA accumulates in a growing loop behind the replisome (Figure 1, Right). As the
replisome moves forward, this loop grows larger from both ends, fed at one end by single-stranded DNA exiting the rear of
the helicase and fed at the other end by newly synthesized double-stranded DNA exiting the rear of the associated
polymerase (Figure 2). When the polymerase reaches the end of one Okasaki fragment it releases the DNA and binds to
the start of the next Okasaki fragment thereby resetting the growing loop back to its original short length. This cycle
repeats down the entire length of the DNA.

As DNA replication progresses the two replication forks with their associated replisomes move farther away from each
other in opposite directions thereby increasing the size of the replication bubble. In prokaryotes, the two replisomes
eventually meet each other halfway from their starting point along the circular chromosome, thereby marking the
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completion of DNA replication (Figure 1). In eukaryotes there would be multiple replisome meeting spots along each
chromosome because of the multiple ORIs (Figure 2, 3).

Once DNA replication is complete each chromosome will have been perfectly duplicated. In eukaryotes each duplicated
chromosome pair remains physically attached at the centromere and the two copies are now referred to as sister
chromatids (Figure 4). In the next module we’ll see how the two sister chromatids are separated from each other to
become two separate chromosomes, with each one ending up in one of the two daughter cells.

Although we focused on DNA replication in prokaryotes throughout this module, we’re now going to switch back to
eukaryotes for the rest of this chapter as we explore the remaining phases of the cell cycle: mitosis and cytokinesis