CELL SIGNALLING, PROLIFERATION & DEATH

A/PROF DONALD WLODKOWIC

GENERAL PRINCIPLES
OF CELL SIGNALLING
CELL-TO-CELL SIGNALLING
FUNDAMENTAL TO ALL ACTIVITIES OF CELLS AND ORGANISMS
Cell signalling is fundamental to all activities of cells. It is
equally important for single cellular organism as well as
multicellular organisms such as us.

No cell lives in isolation on this planet (unless in very specific

laboratory experiments).

In all multicellular organisms, survival depends on an

elaborate intercellular communication network that
coordinates the growth, differentiation, and metabolism of the
multitude of cells in diverse tissues and organs.
CELL-TO-CELL SIGNALLING
FUNDAMENTAL TO ALL ACTIVITIES OF CELLS AND ORGANISMS
ROLE OF THE PLASMA MEMBRANE
RECEIVING AND TRANSMISSION OF SIGNALS (INFORMATION)
SIGNAL TRANSDUCTION
TRANSMITTING THE SIGNAL ACROSS THE PLASMA MEMBRANE
SIGNAL TRANSDUCTION
INVOLVES ALSO A CONVERSION OF THE SIGNAL
SIGNAL TRANSDUCTION
INVOLVES ALSO A CONVERSION OF THE SIGNAL
A process of signal transduction involves a conversion
of the signal from one form into another.

Such process is analogical to conversion of e.g. radio

waves into sound waves.

In cells signal transduction pathways processes of

signal conversion usually involve:
• chemical signalling pathways
• electrical signalling pathways
CELLS ARE GENETICALLY PROGRAMMED
TO RESPOND TO COMBINATIONS OF EXTRACELLULAR SIGNALS
Most of the cells in a complex animal are
programmed to depend on a specific
combination of signals simply to survive.

When deprived of these signals (in a

laboratory culture dish, for example), a
cell activates a suicide program and kills
itself—a process called programmed
cell death, or apoptosis.

Because different types of cells require

different combinations of survival
signals, each cell type is restricted to
different environments in the body.
GENOME INFORMATION IN AN ORGANISM
IS DIFFERENTLY EXPRESSED IN DIFFERENT CELLS
All cells in the same organism have the same genome but the information
contained in the genome is differently expressed in different cells.

As a results it is important to understand that each cell type within a

multicellular organism can react completely differently to the same information
e.g. to the same hormone or a signalling molecule.
RECEPTORS AND LIGANDS
MAIN PLAYERS THAT ENABLE CELL-TO-CELL COMMUNICATION
Target cell is a cell that is receiving the
signal. It responds by means of a
specific protein called a receptor.

Receptor is a protein or protein

complex which specifically binds
the extracellular signal molecule and
then initiates a response in the target
cell.

Ligand is a small molecule that binds to

the receptor. Signal transduction begins
when a receptor binds an extracellular
signalling molecule (a ligand).
BINDING OF A LIGAND TO RECEPTOR
INITIATES THE SIGNAL TRANSDUCTION PATHWAY
ROLES OF INTRACELLULAR ELEMENTS
IN ANY SIGNAL TRANSDUCTION PATHWAY

Intracellular elements of any

signal transduction pathway are
responsible for:
• relaying
• transducing
• amplifying
• integrating
• distributing

the signal incoming from the

receptor
TYPES OF CELL COMMUNICATION
DEPENDING ON DISTANCE THE SIGNAL NEEDS TO TRAVEL
TYPES OF CELL COMMUNICATION
DEPENDING ON DISTANCE THE SIGNAL NEEDS TO TRAVEL
SPEED OF CELL RESPONSES TO SIGNALS
CAN BE VERY RAPID OR VERY SLOW
RAPID CELL RESPONSES TO SIGNALS
REQUIRE PRESENCE OF SIGNALLING PROTEINS IN A CELL
To enable quick changes the proteins that are
elements of the signal transduction pathway need
to be already present in a cell. They are, however,
present in their inactive states e.g. ion channels that
are closed or enzymes that are inactive.

Upon binding of the ligand to the receptor the

signalling cascade quickly turns those protein
ON or modulates their activity (sometimes it actually
switches the proteins off).

One way of understanding such pathway is a series

of switches. In cells we do have special proteins
acting as molecular switches. If you imagine a
series of ON-OFF switches you can grasp those
concepts.
TYPES OF SIGNALLING PATHWAYS
BASED ON THE LOCALISATION OF RECEPTORS
TYPES OF SIGNALLING PATHWAYS
BASED ON THE LOCALISATION OF RECEPTORS
We can distinguish two basic types of cell signalling systems based on
the localisation of receptors in the cell. Those are based on:

1 Cell surface receptors - cell surface receptors are transmembrane

proteins on the target cell surface. When they bind
a hydrophilic extracellular signal molecule, they become activated and
generate a cascade of intracellular signals that alter the behaviour of the cell.

2 Intracellular receptors - intracellular receptors as their name suggests

are inside the target cell, and the signal molecule has to enter the cell to
activate them: this requires that the signal molecules be sufficiently small
and hydrophobic to diffuse across the plasma membrane. As you might
remember from Week 3 topic about cell membranes to pass a lipid bilayer
ligands for such intracellular receptors must be small hydrophobic
compounds such as steroid hormones.
 SIGNALLING THROUGH
CELL-SURFACE RECEPTORS
SIGNALLING VIA CELL SURFACE RECEPTORS
IS BASED ON HYDROPHILIC LIGANDS THAT DO NOT PASS MEMBRANES

All cell surface receptors

bind hydrophilic extracellular
signal molecules.

Such hydrophilic ligands are usually

proteins or glycoproteins
that would not be able to pass
through the cell membrane.

When cell surface receptors become

activated and generate a cascade
of intracellular signals that alter the
behaviour of the cell.
ACTIVATION OF CELL SURFACE RECEPTORS
GENERATES A CASCADE OF INTRACELLULAR SIGNALS
CLASSES OF CELL SURFACE RECEPTORS
ION CHANNEL-, G-PROTEIN-, ENZYME-COUPLED RECEPTORS
G-PROTEIN-COUPLED RECEPTORS
ACT THOUGH A SMALL TRIMERIC G-PROTEIN
G-protein-coupled receptors (GPCR) act indirectly to regulate the activity of a separate plasma-
membrane-bound target protein, which can be either an enzyme or an ion channel.

The interaction between the receptor and this target protein is mediated by a third protein
called GTP-binding protein (often referred to as G protein). G-proteins act as signal relays that can
activate or inhibit an enzyme or an ion channel at the plasma membrane thus initiating an intracellular
signalling cascade. All of the G-protein-coupled receptors (GPCRs) belong to a large family
of seven-pass transmembrane proteins.
ENZYME-COUPLED RECEPTORS
ACT AS ENZYMES OR ARE ASSOCIATED WITH ENZYMES
Enzyme-coupled receptors act as enzymes or are associated with enzymes. Enzyme-coupled
receptors are formed single-pass transmembrane proteins that have their ligand-binding site
outside the cell and their catalytic or enzyme-binding site inside.

The great majority of enzyme-coupled receptors are protein kinases, or are associated with protein
kinases, and their activation causes the phosphorylation of specific sets of proteins in the target cell.
 PHOSPHORYLATION CASCADES
CONTROL SIGNALLING PROTEINS BY PHOSPHORYLATION
Many of the signalling proteins are controlled by phosphorylation. Many of the
intracellular signalling proteins are themselves enzymes that are called protein
kinases.

Phosphorylation cascade occurs when one protein kinase, activated by

phosphorylation, phosphorylates the next protein kinase in the sequence, and so
on, relaying the signal onward and, in the process, amplifying it and sometimes
spreading it to other signaling pathways.

Two main types of protein kinases operate as intracellular signalling proteins:

• Serine/threonine kinases. The great majority are serine/threonine kinases,
which are enzyme that phosphorylate proteins on amino acids such as serine and
(less often) threonine.
• Tyrosine kinases are enzymes that phosphorylate proteins on amino acid
tyrosine.
PHOSPHORYLATION CASCADES
CONTROL SIGNALLING PROTEINS ARE BY PHOSPHORYLATION
 SECOND MESSENGERS
IN SIGNAL TRANSDUCTION PATHWAYS
The small intracellular signalling molecules
are called second messengers.

The first messengers are actually the

extracellular signals.

Second messengers are generated in

large numbers in response to receptor
activation and rapidly diffuse away from
the receptor, broadcasting the signal to
other parts of the cell.

Examples of second messengers include:

• cyclic AMP
• ions of calcium (Ca2+)
• diacylglycerol
• inositol triphosphate (IP3)
SECOND MESSENGERS
IN SIGNAL TRANSDUCTION PATHWAYS
INTRACELLULAR SIGNALLING PROTEINS
CAN ACT AS MOLECULAR SWITCHES AND MOLECULAR TIMERS
The molecular switches fall into TWO main classes that operate in different ways:

1 Class 1 - Signalling via direct phosphorylation. This is the largest class that
consists of proteins that are activated or inactivated by phosphorylation. For these
proteins, the switch is thrown in one direction by a protein kinase, which adds one or
more phosphate groups to the signalling protein, and in the other direction by a
protein phosphatase, which removes the phosphate groups from the protein. It is
estimated that one-third of the proteins in a eukaryotic cell are phosphorylated at any
given time.

2 Class 2 - Signalling via GTP-binding proteins. These switch between an active

state when GTP is bound and an inactive state when GDP is bound. Once activated,
they have intrinsic GTPase activity and shut themselves off by hydrolyzing their bound
GTP to GDP. GTP-binding proteins are large trimeric GTP-binding proteins (also called G
proteins). Trimeric means that the are composed of three separate parts. They relay the
signals from G-protein-linked receptors.
INTRACELLULAR SIGNALLING PROTEINS
CAN ACT AS MOLECULAR SWITCHES AND MOLECULAR TIMERS
 SIGNALLING THROUGH
INTRACELLULAR RECEPTORS
INTRACELLULAR RECEPTOR LIGANDS
ARE SMALL HYDROPHOBIC MOLECULES
A number of small
hydrophobic signal molecules
can actually diffuse directly
across the plasma membrane
of target cells and bind to
intracellular receptor
proteins.

This is in very stark contrast

to cell surface receptors that
bind hydrophilic ligands that
cannot penetrate the barrier of
phospholipid bilayer.
INTRACELLULAR RECEPTOR LIGANDS
INCLUDE STEROID HORMONES SUCH AS CORTISOL
Signal molecules that can diffuse
through the plasma membrane
include steroid hormones,
thyroid hormones, retinoids, and
vitamin D. Although they differ
greatly from one another in both
chemical structure and function,
they all act by a similar
mechanism.

Steroid hormones—which
include cortisol and the steroid
sex hormone testosterone are
all made from cholesterol.
INTRACELLULAR RECEPTORS
ARE TRANSCRIPTION FACTORS REGULATING GENE EXPRESSION
The inactive receptors are bound to inhibitory protein complexes
but can bind steroid hormones in the cytosol.

The binding of a ligand e.g. cortisol alters the conformation of the receptor
protein, causing the inhibitory complex to dissociate.
INTRACELLULAR RECEPTORS
ARE TRANSCRIPTION FACTORS REGULATING GENE EXPRESSION

Upon the conformational

change the freed activated
receptor subsequently
translocates from the cytosol
to the nucleus.

In nucleus the receptor acts as

a transcription
factor, activating a
transcription of special sets of
genes. This process leads
to regulation of gene expression.
CELL PROLIFERATION
DYNAMICS OF A CELL
BEST UNDERSTOOD BY STUDYING THE LIFE CYCLE OF A CELL
Two critical periods in the life
cycle of a cell:
➤ cell growth, a preparation
period during which proteins
are made and DNA is
replicated in anticipation of
cell division
➤ cell division, that combines
karyokinesis and cytokinesis
when a cell divides into two
daughter cells.
CELL DIVISION AND GENOMES
PASSED ONTO THE DAUGHTER CELLS
THE CELL CYCLE
EUKARYOTIC CELL CYCLE
IS VERY COMPLEX AND MUCH SLOWER THAN IN PROKARYOTES
Most growing plant and animal cells take
10 – 20 hours to double in number, and
some duplicate at a much slower rate.
Many cells in adult animals, such as
nerve cells and striated muscle cells, do
not divide at all. They have temporarily
exited from the cell cycle after mitosis and
entered a “paused or quiescent” state
called G0.
Because eukaryotic cells are larger and
more complex than prokaryotic cells, a
specialized mechanism coordinates their
replication of genomic DNA, distribution
of chromosomes, and cell division.
EUKARYOTIC CELL CYCLE
IS DIVIDED INTO SEVERAL CHARACTERISTIC PHASES
INTERPHASE
INTERPHASE
DENOTES PREPARATION OF CELL FOR DIVISION
INTERPHASE
COMPRISES OF G1, S AND G2 PHASES
Interphase is a part of the cell
cycle that enables cellular
macromolecules to be
synthesised throughout its
duration, so the cell roughly
doubles its mass.
Interphase comprises of sub-
phases called:
➤ G1 (Gap 1) phase
➤ S (Synthesis) phase
➤ G2 (Gap 2) phase
INTERPHASE
G1 PHASE
During G1 phase, the cell grows in
size and synthesizes mRNA and
proteins (e.g. histones) that are
required for DNA synthesis.
Once the required proteins and growth
are complete, the cell enters the next
phase of the cell cycle, S phase.
The G1 phase is particularly
important in the cell cycle because it
determines whether a cell commits to
division or to leaving the cell cycle.
If a cell is signalled to remain undivided, instead of moving onto the S phase, it will
leave the G1 phase and move into a state of dormancy called the G0 phase. Most
nonproliferating vertebrate cells will enter the G0 phase
INTERPHASE
S PHASE
The main role of S phase is in DNA replication which requires 10–12
hours and occupies about half of the cell-cycle time in a typical
mammalian cell.
INTERPHASE
G2 PHASE
G2 phase follows the successful
completion of S phase, during which
the cell’s DNA is replicated and
directly precedes the mitosis.
During G2 the cell is prepared for the
mitotic (M) phase, when the genetic
material is evenly partitioned and the cell
divides.
In vertebrate cells, the G2/M DNA
damage checkpoint consists of an
arrest of the cell in G2 just before
mitotic entry in response to genotoxic
stress (such as UV radiation, oxidative
stress, DNA intercalating agents, etc.).
THE ROLE OF GAP PHASES
ENABLE CELL TO EVALUATE INTERNAL AND EXTERNAL ENVIRONMENT
THE ROLE OF GAP PHASES
ENABLE CELL TO EVALUATE INTERNAL AND EXTERNAL ENVIRONMENT
THE CONTROL OF CELL CYCLE
REGULATION OF THE CELL CYCLE
INVOLVES A SERIES OF REGULATORY CHECKPOINTS
Progress along the cycle
is controlled at a series
of special checkpoints.
Each checkpoint monitor
the status of a cell, for
instance, the internal
amount of DNA or the
presence of extracellular
nutrients.
When certain conditions
are met, the cell proceeds
to the next checkpoint.
REGULATION OF THE CELL CYCLE
INVOLVES CYCLINS AND CYCLIN-DEPENDENT KINASES
The complex events of the eukaryotic cell
cycle are regulated by a small number of
heterodimeric protein kinases.
•catalytic subunits are called cyclin-
dependent kinases (Cdks) because they
have no kinase activity unless they are
associated with a regulatory part called cyclin
•regulatory (control) subunits called
cyclins the concentrations of the regulatory
subunits, increase and decrease in phase with
the cell cycle.
CYCLIN CONCENTRATIONS
ARE REGULATED BY SYNTHESIS AND PROTEOLYTIC DEGRADATION
CYCLINS SPECIFIC FOR CELL CYCLE PHASES
ACT AS “ON” SWITCHES

The concentration of various regulatory cyclins that are specific to

certain phase of the cell cycle varies as the cell progresses through its
cell cycle. In short we might say that cyclins are used as "ON" switches
that initiate transition between the consecutive phases of the cell cycle.
CYCLIN CONCENTRATIONS
ARE REGULATED BY SYNTHESIS AND PROTEOLYTIC DEGRADATION
CYCLIN ARE DEGRADED
IN A PROTEASOME - A CELL’S PROTEIN SHREDDER
Proteasomes are special protein complexes which degrade unneeded or
damaged proteins by proteolysis, a chemical reaction that breaks peptide
bonds. Enzymes that help such reactions are called proteases.
CDK COMPLEXES
CAN BE ALSO REGULATED BY PHOSPHORYLATION
M PHASE
M PHASE
COMPRISES OF KARYOKINESIS AND CYTOKINESIS
In M phase chromosome segregation and cell division occur. The copies of
chromosomes (sister chromatids) are distributed to opposite ends of the cell
during M phase. This requires much less time (less than an hour in a
mammalian cell). M phase involves a series of dramatic events that begin
with nuclear division (karyokinesis) and culminate in cytokinesis.
KARYOKINESIS
THE PARTITIONING OF THE GENOME

Karyokinesis (mitosis) is the

mechanism in eukaryotes for
partitioning the genome equally at cell
division.
To accomplish this complex task, plant
and animal cells build a specialized
machine, called the mitotic apparatus,
which captures the chromosomes and
then pushes and pulls them to
opposite sides of the dividing cell.
STAGES OF MITOSIS
5 MAIN STAGES OF KARYOKINESIS
PROPHASE
1ST STAGE OF KARYOKINESIS
PROMETAPHASE
2ND STAGE OF KARYOKINESIS
METAPHASE
3RD STAGE OF KARYOKINESIS
ANAPHASE
4TH STAGE OF KARYOKINESIS
TELOPHASE
5TH STAGE OF KARYOKINESIS
DISASSEMBLY AND RE-ASSEMBLY
OF THE NUCLEUS DURING MITOSIS
REPLICATION OF A CENTROSOME
IS CRUCIAL FOR MITOTIC SPINDLE FORMATION
MOVEMENT OF CHROMOSOMES
DEPENDS ON THE MICROTUBULES AND MOLECULAR MOTORS
CHROMOSOMES
ARE ATTACHED AT THE REGION CALLED KINETOCHORE
MOVEMENT OF CHROMOSOMES
DEPENDS ON THE MICROTUBULES AND MOLECULAR MOTORS
ROLE OF CYTOSKELETON
IN CELL DIVISION
ROLE OF CYTOSKELETON
IN CELL DIVISION
CYTOKINESIS
IN ANIMAL CELLS
CYTOKINESIS IN ANIMAL CELLS
REQUIRES FORMATION OF A CONTRACTILE RING
CYTOKINESIS IN ANIMAL CELLS
IS MOSTLY SYMMETRICAL
CYTOKINESIS IN PLANT CELLS
UTILISES A STRUCTURE CALLED PHRAGMOPLAST
CYTOKINESIS IN PLANT CELLS
UTILISES A STRUCTURE CALLED PHRAGMOPLAST
PHRAGMOPLAST
ENABLES PLANT CELLS TO FORM NEW CELL WALLS
The phragmoplast is a complex assembly of microtubules (MTs),
microfilaments (MFs), and endoplasmic reticulum (ER) elements, that
assemble in two opposing sets perpendicular to the plane of the future cell
plate during anaphase and telophase.

The vesicles derived from the Golgi apparatus are all part of the
phragmoplast. The vesicles from Golgi apparatus are filled with cell-wall
material and fuse to form the growing new cell wall, which grows outward
to reach the plasma membrane and original cell wall at the site determined
earlier by the preprophase band.
CELL DEATH
CELL DIVISION AND CELL DEATH
AN INTERTWINED RELATIONSHIP OF LIFE AND DEATH
We can not talk about cell division and cell cycle without mentioning cell death.
Unchecked cell growth and multiplication of cells produce a mass of cells
commonly known as a tumor (cancer).
Any cell in our body can be directed to commit suicide when an essential factor is
removed from the extracellular environment or when an internal "death" signals are
activated. Thus, the default state of the cell is to remain alive.
ACCIDENTAL VS PROGRAMMED
DIFFERENT TYPES OF CELL DEMISE
APOPTOSIS
MITOCHONDRIAL VS DEATH RECEPTOR PATHWAYS
APOPTOSIS
MITOCHONDRIAL VS DEATH RECEPTOR PATHWAYS
Mitochondrial(or intrinsic) pathway is
engaged by cellular stress or developmental
cues that alter the expression and/or function
of proteins of the Bcl-2 family. This pathway
directly causes mitochondrial outer mem-
brane permeabilization (MOMP), releasing
proteins of the mitochondrial inter-membrane
space into the cytosol. Proteins released to the
cytosol upon MOMP include cytochrome c,
which binds to the cytosolic protein APAF1
inducing its oligomerization into the
apoptosome. The apoptosome activates the
characteristic cascade of the executioner
caspases (hence caspase-dependent apoptosis).
APOPTOSIS
MITOCHONDRIAL VS DEATH RECEPTOR PATHWAYS
Death receptor (or extrinsic) pathway is
engaged is engaged when a subset of the
tumor necrosis factor (TNF) receptor family
(the death receptors) are ligated. The
intracellular region of the death receptor, upon
ligation, recruits the adapter molecule FADD,
which in turn recruits the initiator caspase,
caspase-8. Bound caspase-8 then recruits
additional caspase-8 molecules, which are
activated by dimerization. The active caspase-8
cleaves and thereby activates the executioner
caspase-3 and caspase-7.
.
CASPASES
PROTEOLYTIC ENZYMES THAT EXECUTE THE APOPTOSIS
CASPASES
PROTEOLYTIC ENZYMES THAT EXECUTE THE APOPTOSIS
APOPTOSIS
FINAL STAGES SEEN IN A SCANNING ELECTRON MICROSCOPE
ROLE OF APOPTOSIS
IN DEVELOPMENT & TISSUE REMODELLING
APOPTOSIS
CRUCIAL ROLE IN HOMEOSTASIS AND PATHOGENESIS

Programmed cell
death plays the very
important role of
population control by
balancing cell growth
and multiplication.
In addition, cell death
also eliminates
unnecessary or
pathologically changed
cells.
ROLE OF APOPTOSIS
IN IMMUNE SURVEILLANCE AND CANCER CONTROL
DISREGULATED APOPTOSIS AND CANCERS
APOPTOSIS IS FUNDAMENTAL FOR KEEPING CANCER IN CHECK
APOPTOSIS
METHODS OF DETECTION & ANALYSIS IN THE LABORATORY
APOPTOSIS
METHODS OF DETECTION & ANALYSIS IN THE LABORATORY
APOPTOSIS
METHODS OF DETECTION & ANALYSIS IN THE LABORATORY
MANY WAYS TO DIE
BEYOND APOPTOSIS AND ONCOSIS
THANK YOU