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Cells
Volume 7
Issue 6
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Cells, Volume 7, Issue 6 (June 2018) – 18 articles

Cover Story (view full-size image): Single cell analysis provides an approach to directly link external stimulation of an individual cell to molecular changes in that same cell. Nanopipette technology can provide multiplexed, single cell analysis over time. The core technology employs a nanopipette instrument, which is a hollow quartz needle with a tip opening of less than 100 nm. By employing scanning ion conductance microscopy, changes in ion current through the nanopipette tip can be used to find cell surfaces using a proximity effect known as “current squeezing”, which Pourmand’s team have previously described. View the paper here.
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35 pages, 1252 KiB  
Review
Endoplasmic Reticulum Stress in Metabolic Disorders
by Rose Ghemrawi, Shyue-Fang Battaglia-Hsu and Carole Arnold
Cells 2018, 7(6), 63; https://doi.org/10.3390/cells7060063 - 19 Jun 2018
Cited by 153 | Viewed by 15030
Abstract
Metabolic disorders have become among the most serious threats to human health, leading to severe chronic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease, as well as cardiovascular diseases. Interestingly, despite the fact that each of these diseases has [...] Read more.
Metabolic disorders have become among the most serious threats to human health, leading to severe chronic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease, as well as cardiovascular diseases. Interestingly, despite the fact that each of these diseases has different physiological and clinical symptoms, they appear to share certain pathological traits such as intracellular stress and inflammation induced by metabolic disturbance stemmed from over nutrition frequently aggravated by a modern, sedentary life style. These modern ways of living inundate cells and organs with saturating levels of sugar and fat, leading to glycotoxicity and lipotoxicity that induce intracellular stress signaling ranging from oxidative to ER stress response to cope with the metabolic insults (Mukherjee, et al., 2015). In this review, we discuss the roles played by cellular stress and its responses in shaping metabolic disorders. We have summarized here current mechanistic insights explaining the pathogenesis of these disorders. These are followed by a discussion of the latest therapies targeting the stress response pathways. Full article
(This article belongs to the Special Issue Cellular Stress Response in Health and Disease)
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<p>ER stress and the UPR. IP3R receptor and calcium pump SERCA are responsible for the efflux and influx of calcium in ER, respectively. Intracellular calcium dysregulation induces ER stress. ER stress induces the UPR. Unfolded or misfolded proteins are recognized by Bip, which then releases itself from PERK, IRE1, and ATF6, leading to their activation. The activation of PERK involves its homodimer formation and autophorsphorylation. It subsequently phosphorylates eIF2α, which in turn attenuates general translation but facilitates the translation of ATF4, thus increasing its protein level. ATF4 is a transcription factor that activates the transcription of CHOP. Phosphorylated p38 MAPK phosphorylates CHOP, which then triggers the transcription of apoptotic genes. Similar to PERK, IRE1α activates after the formation and autophosphorylation of IRE1α homodimers. This activates IRE1α RNase activity. IRE1α then splices <span class="html-italic">Xbp1</span>, removing 26 nucleotides from its transcript. Then, the XBP1s spliced form of XBP1 acts as a transcription factor for chaperones, as well as genes involved in ERAD and lipid biosynthesis. ATF6 activation involves its release from Bip, and its translocation into the Golgi, where it undergoes cleavage by S1P/S2P proteases. This allows its nuclear translocation and subsequent transcriptional activation of chaperones, XBP1, CHOP, and ERAD genes. Abbreviations: IP3R (inositol 1,4,5-trisphosphate(IP3R) receptor).</p> Full article ">Figure 2
<p>ER and the mitochondria function together as sensors of nutrient excess and integrators of the response to metabolic stress. Reactive oxygen species (ROS) are free radicals that are generated as a by-product of cellular metabolism from FA and glucose in the mitochondria. FA enhance MAM; thus, excessive cytoplasmic calcium enters in mitochondria and ER, triggering changes in mitochondrial pH and ROS production. This alters mitochondrial membrane potential and opens pores, thus releasing cytochrome c. Several calcium-dependent proteins and kinases are then activated, triggering apoptosis. ROS perturb the redox status of ER lumen and thus inhibit protein folding. The ER-associated protein NADPH oxidase 4 (NOX4) is implicated in ROS generation during disulfide bond formation for proper protein folding. Excess ROS cause oxidative stress, which induces the 3 branches of the UPR. The result of the UPR activation is inflammation and, upon sustained and/or intense stress, apoptosis. FA also disrupt SERCA activity and perturb ER calcium homeostasis. The mTORC1 complex is an important sensor of bioenergetic status and nutrient excess, and induces ER stress by unclear mechanisms. PERK-mediated activation of ATF4 induces NRF2, a transcription factor responsible for antioxidant cell response. Abbreviations: cyt c, cytochrome c (cyt c); Ox. stress, oxidative stress (Ox. Stress); MAM, mitochondria-associated membranes (MAM); mTORC1, mammalian target of rapamycin complex 1 (mTORC1); NOX4, NADPH oxidase 4 (NOX4); ROS, Reactive oxygen species (ROS); SERCA, sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Sharp arrows, activators; bar-ended arrows, inhibitors.</p> Full article ">Figure 3
<p>Induction and consequences of ER stress in insulin resistance and diabetes. In diabetes, lipotoxicity and glucotoxicity induce ER stress in several cell types. Inflammation is another inducer of ER stress. In beta cells, intense insulin production leads to misfolded insulin and hIAPP aggregates, which induce ER stress. ER stress triggers various responses, including inflammation, IR, apoptosis, decrease of insulin secretion, and increase of gluconeogenesis and lipogenesis, depending on the considered cell type.</p> Full article ">Figure 4
<p>ER stress and the UPR in insulin resistance and diabetes. Insulin resistance and diabetes involve dysregulations in multiple organs, and ER stress participates to all these dysregulations. Excessive dietary FA and glucose induce ER stress in the brain, beta cells of Langerhans islets, myocytes, hepatocytes, and adipocytes. In the brain, this triggers insulin resistance and leptin resistance, with the latter enhancing appetite and thus excessive nutrient intake. In hepatocytes, FA-induced ER stress induces the three branches of the UPR. The PERK arm induces VLDLR and CHOP; the IRE1 arm increases lipogenesis and the CREBH arm enhances neoglucogenesis. In myocytes, this induces the IRE1 arm of the UPR, which deregulates the insulin receptor signaling and thus leads to insulin resistance. In adipocytes, FA and glucose trigger the IRE1 and PERK pathways, which induce, respectively, insulin resistance via JNK induction, and adipokines, TNFα, and IL-6 secretion. These adipokines and cytokines are released into the bloodstream and trigger ER stress in beta cells. FA, IAPP, and insulin overproduction are other triggers of ER stress in this cell type. Consequences of ER stress in beta cell are apoptosis through the PERK/eIF2α/ATF4/CHOP pathway and decreased GSIS via GLUT2 repression. Abbreviations: ERS, ER stress; GSIS, Glucose-Stimulated Insulin Secretion; IR, Insulin Resistance.</p> Full article ">Figure 5
<p>Targeting ER stress and the UPR with chemical and pharmacological drugs in metabolic diseases. TUDCA and PBA are chemical chaperones that improve protein folding in a non-specific manner. Valproate and BIX Bix are two small molecules, BIP Bip inducers. Verapamil is a calcium channel blocker, which includes the calcium release channel IP3R. CDN1163 is a small allosteric activator of SERCA2b. GSK2606414 and GSK2656157 are PERK inhibtors. Salubrinal is a small molecule, which reduces dephosphorylation of eIF2α. Ursolic acid and tomatidine are inhibitors of ATF4. SB203580 is a specific inhibitor of the p38 MAP kinase, which phosphorylates CHOP. STF-083010 and 4µ8C are inhibitors of IRE1 RNase activity. The small molecule 2-[5-[1-(4-chlorophenoxy)ethyl]-4-phenyl-4<span class="html-italic">H</span>-1,2,4-triazol-3-yl]sulfanyl-<span class="html-italic">N</span>-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1<span class="html-italic">H</span>-pyrazol-4-yl)acetamide (TSPA) is a small allosteric activator, which functions as an ATF6α translocation inducer. Abbreviations: inositol 1,4,5-trisphosphate(IP3R) receptorIP3R (inositol 1,4,5-trisphosphate) receptor. Grey boxes contain the name of the drugs (sharp arrows, activators; bar-ended arrows, inhibitors).</p> Full article ">
15 pages, 281 KiB  
Review
Role of the TRPM4 Channel in Cardiovascular Physiology and Pathophysiology
by Chen Wang, Keiji Naruse and Ken Takahashi
Cells 2018, 7(6), 62; https://doi.org/10.3390/cells7060062 - 15 Jun 2018
Cited by 38 | Viewed by 6151
Abstract
The transient receptor potential cation channel subfamily M member 4 (TRPM4) channel influences calcium homeostasis during many physiological activities such as insulin secretion, immune response, respiratory reaction, and cerebral vasoconstriction. This calcium-activated, monovalent, selective cation channel also plays a key role in cardiovascular [...] Read more.
The transient receptor potential cation channel subfamily M member 4 (TRPM4) channel influences calcium homeostasis during many physiological activities such as insulin secretion, immune response, respiratory reaction, and cerebral vasoconstriction. This calcium-activated, monovalent, selective cation channel also plays a key role in cardiovascular pathophysiology; for example, a mutation in the TRPM4 channel leads to cardiac conduction disease. Recently, it has been suggested that the TRPM4 channel is also involved in the development of cardiac ischemia-reperfusion injury, which causes myocardial infarction. In the present review, we discuss the physiological function of the TRPM4 channel, and assess its role in cardiovascular pathophysiology. Full article
(This article belongs to the Special Issue TRP Channels in Health and Disease)
10 pages, 3648 KiB  
Article
Expression Profiling of the Transient Receptor Potential Vanilloid (TRPV) Channels 1, 2, 3 and 4 in Mucosal Epithelium of Human Ulcerative Colitis
by Theodoros Rizopoulos, Helen Papadaki-Petrou and Martha Assimakopoulou
Cells 2018, 7(6), 61; https://doi.org/10.3390/cells7060061 - 15 Jun 2018
Cited by 38 | Viewed by 5344
Abstract
The Transient Receptor Potential (TRP) family of selective and non-selective ion channels is well represented throughout the mammalian gastrointestinal track. Several members of the Transient Receptor Potential Vanilloid (TRPV) subfamily have been identified in contributing to modulation of mobility, secretion and sensitivity of [...] Read more.
The Transient Receptor Potential (TRP) family of selective and non-selective ion channels is well represented throughout the mammalian gastrointestinal track. Several members of the Transient Receptor Potential Vanilloid (TRPV) subfamily have been identified in contributing to modulation of mobility, secretion and sensitivity of the human intestine. Previous studies have focused on the detection of TRPV mRNA levels in colon tissue of patients with inflammatory bowel disease (IBD) whereas little information exists regarding TRPV channel expression in the colonic epithelium. The aim of this study was to evaluate the expression levels of TRPV1, TRPV2, TRPV3 and TRPV4 in mucosa epithelial cells of colonic biopsies from patients with ulcerative colitis (UC) in comparison to colonic resections from non-IBD patients (control group). Immunohistochemistry, using specific antibodies and quantitative analyses of TRPV-immunostained epithelial cells, was performed in semi-serial sections of the samples. TRPV1 expression was significantly decreased whereas TRPV4 expression was significantly increased in the colonic epithelium of UC patients compared to patients in the control group (p < 0.05). No significant difference for TRPV2 and TRPV3 expression levels between UC and control specimens was detected (p > 0.05). There was no correlation between TRPV channel expression and the clinical features of the disease (p > 0.05). Further investigation is needed to clarify the role of TRPV channels in human bowel inflammatory response. Full article
(This article belongs to the Special Issue TRP Channels in Health and Disease)
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<p>TRPV1 immunolocalization in UC and control non-IBD samples (<b>A</b>–<b>E</b>) and TRPV3 nerve fiber immunolabeling in UC (<b>F</b>). (<b>A</b>) Renal tissue sections were used as positive control for TRPV1-immunostaining; (<b>B</b>) Strong cytoplasmic TRPV1 immunoreactivity in mucosal epithelium of control group. Note TRPV1-immunostained cells in lamina propria; (<b>C</b>) Cells in enteric nervous system display strong TRPV1 immunopositivity. Furthermore, endothelial cells are TRPV1-immunoreactive (arrows); (<b>D</b>) Strong cytoplasmic TRPV1 immunostaining in a few superficial mucosa cells of this UC specimen; (<b>E</b>) Heterogeneity in TRPV1 in epithelium of UC sample. Note TRPV1-immunonegative mucosa cells nearby to TRPV1-immunopositive mucosa cells (LI = 50). (<b>F</b>) TRPV3 immunoreactivity in nerve fibers in UC. Counterstain, hematoxylin; original magnification, ×400 (<b>A</b>–<b>D</b>,<b>F</b>), ×200 (<b>E</b>); scale bar, 50 μm.</p> Full article ">Figure 2
<p>Panel presenting expression patterns of TRPV2 (<b>B</b>,<b>C</b>) and TRPV3 (<b>D</b>,<b>F</b>) in UC and control samples; (<b>A</b>) Ophthalmic pterygium tissue samples were used as positive controls for TRPV2 immunoreactivity; (<b>B</b>) TRPV2-immunostaining in intestinal epithelial cells of control colon. ((<b>B</b>), insert) Cells in enteric nervous system display strong TRPV2-immunopositivity; (<b>C</b>) Moderate cytoplasmic TRPV2 staining in numerous mucosa cells in UC specimen. Several TRPV2-immunopositive cells are observed in lamina propria cells; (<b>D</b>) Renal tissue sections were used as positive control for TRPV3-immunostaining; (<b>E</b>) Aberrant cytoplasmic TRPV3-immunostaining in epithelial cells of control sample. Note the strong-immunostained smooth muscle cells in muscularis mucosa; (<b>F</b>) Cytoplasmic expression of TRPV3 in epithelium and muscularis mucosa of UC sample. Counterstain, hematoxylin; original magnification, ×400; scale bar, 50 μm.</p> Full article ">Figure 3
<p>Panel depicting the cellular distribution of TRPV4 in UC and control intestine specimens. (<b>A</b>) Renal tissue sections were used as positive control for TRPV4-immunostaining. (<b>B</b>) Weak TRPV4-immunostaining in intestinal epithelial cells of control colon. (<b>C</b>) Granular cytoplasmic TRPV4-immunoexpression is identified in the cytoplasm of numerous mucosa cells in UC. (<b>D</b>) Nuclei of mucosa cells display TRPV4 immunostaining in this UC sample. (<b>E</b>,<b>F</b>) Strong granular cytoplasmic TRPV4 immunolocalization in superficial mucosa cells and goblet cells whereas there are mucosa cells with weak immunostaining of UC samples. ((<b>E</b>), insert) Immunostaining is absent in negative control sections. Counterstain, hematoxylin; original magnification, ×400; scale bar, 50 μm.</p> Full article ">Figure 4
<p>Comparison of TRPV channel expression in mucosa epithelial cells of UC and non-IBD control samples. Significant differences between UC and non-IBD group for TRPV1 and TRPV4 expression was detected (<span class="html-italic">p</span> &lt; 0.05). No significant difference was observed regarding TRPV2 and TRPV3 expression (<span class="html-italic">p</span> &gt; 0.05).</p> Full article ">
19 pages, 1054 KiB  
Review
Real-Time Imaging of Retinal Ganglion Cell Apoptosis
by Timothy E. Yap, Piero Donna, Melanie T. Almonte and Maria Francesca Cordeiro
Cells 2018, 7(6), 60; https://doi.org/10.3390/cells7060060 - 15 Jun 2018
Cited by 34 | Viewed by 7094
Abstract
Monitoring real-time apoptosis in-vivo is an unmet need of neurodegeneration science, both in clinical and research settings. For patients, earlier diagnosis before the onset of symptoms provides a window of time in which to instigate treatment. For researchers, being able to objectively monitor [...] Read more.
Monitoring real-time apoptosis in-vivo is an unmet need of neurodegeneration science, both in clinical and research settings. For patients, earlier diagnosis before the onset of symptoms provides a window of time in which to instigate treatment. For researchers, being able to objectively monitor the rates of underlying degenerative processes at a cellular level provides a biomarker with which to test novel therapeutics. The DARC (Detection of Apoptosing Retinal Cells) project has developed a minimally invasive method using fluorescent annexin A5 to detect rates of apoptosis in retinal ganglion cells, the key pathological process in glaucoma. Numerous animal studies have used DARC to show efficacy of novel, pressure-independent treatment strategies in models of glaucoma and other conditions where retinal apoptosis is reported, including Alzheimer’s disease. This may forge exciting new links in the clinical science of treating both cognitive and visual decline. Human trials are now underway, successfully demonstrating the safety and efficacy of the technique to differentiate patients with progressive neurodegeneration from healthy individuals. We review the current perspectives on retinal ganglion cell apoptosis, the way in which this can be imaged, and the exciting advantages that these future methods hold in store. Full article
(This article belongs to the Special Issue Innovative Methods to Monitor Single Live Cells)
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<p>DARC imaging highlighting apoptosing retinal ganglion cells (<b>a</b>) using intravitreal ANX776 in a rat model of glaucoma following episcleral vein injections of hypertonic saline; (<b>b</b>) using intravenous ANX776 in a human glaucoma patient shown to have progressive disease.</p> Full article ">Figure 2
<p>Potential RGC-neuroprotective agents and their targeted pathogenic processes, some of which have been studied with DARC imaging. (ROS: Reactive Oxygen Species).</p> Full article ">Figure 3
<p>Lifespan-adjusted projection of DARC counts extrapolated from a rodent model of glaucoma, and superimposed onto a human disease course over 30 years. This demonstrates potential for diagnosis of early disease using DARC.</p> Full article ">
16 pages, 754 KiB  
Review
Unconventional Secretion and Intercellular Transfer of Mutant Huntingtin
by Bor Luen Tang
Cells 2018, 7(6), 59; https://doi.org/10.3390/cells7060059 - 14 Jun 2018
Cited by 21 | Viewed by 5864
Abstract
The mechanism of intercellular transmission of pathological agents in neurodegenerative diseases has received much recent attention. Huntington’s disease (HD) is caused by a monogenic mutation in the gene encoding Huntingtin (HTT). Mutant HTT (mHTT) harbors a CAG repeat extension which encodes an abnormally [...] Read more.
The mechanism of intercellular transmission of pathological agents in neurodegenerative diseases has received much recent attention. Huntington’s disease (HD) is caused by a monogenic mutation in the gene encoding Huntingtin (HTT). Mutant HTT (mHTT) harbors a CAG repeat extension which encodes an abnormally long polyglutamine (polyQ) repeat at HTT’s N-terminus. Neuronal pathology in HD is largely due to the toxic gain-of-function by mHTT and its proteolytic products, which forms both nuclear and cytoplasmic aggregates that perturb nuclear gene transcription, RNA splicing and transport as well cellular membrane dynamics. The neuropathological effects of mHTT have been conventionally thought to be cell-autonomous in nature. Recent findings have, however, indicated that mHTT could be secreted by neurons, or transmitted from one neuronal cell to another via different modes of unconventional secretion, as well as via tunneling nanotubes (TNTs). These modes of transmission allow the intercellular spread of mHTT and its aggregates, thus plausibly promoting neuropathology within proximal neuronal populations and between neurons that are connected within neural circuits. Here, the various possible modes for mHTT’s neuronal cell exit and intercellular transmission are discussed. Full article
(This article belongs to the Special Issue Unconventional Protein Secretion in Development and Disease)
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<p>A schematic diagram illustrating the various modes of mutant Huntingtin (mHTT) unconventional secretion and intercellular propagation. Secretion from one neuron (I) could occur via exosomes when luminal vesicles from the multivesicular body (MVB) fuse with the plasma membrane. These exosomes could be taken up endocytically by another neuron (II). Secretion could also occur via a lysosome-based mechanism, with the release of non-vesicular mHTT. Interneuronal transfer, particularly between neurons (I and III) connected within a neural circuit, could occur via vesicles generated at the synaptic terminals. Intercellular transfer of mHTT aggregates could also occur via tunneling nanotubes (TNTs). N—nucleus. See text for more details.</p> Full article ">
12 pages, 1898 KiB  
Article
Effect of Notch and PARP Pathways’ Inhibition in Leukemic Cells
by Luka Horvat, Mariastefania Antica and Maja Matulić
Cells 2018, 7(6), 58; https://doi.org/10.3390/cells7060058 - 14 Jun 2018
Cited by 7 | Viewed by 4063
Abstract
Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the [...] Read more.
Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors. Full article
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<p>Characterization of the Notch signaling pathway in leukemic cells. Notch signaling activity in T cell line (Jurkat), B chronic lymphocytic cell line (CLL) and B cell precursor leukemia cell line (697) was analyzed by Western blot. FL: full-length; NTM: transmembrane/intracellular region; ACTB: β-actin.</p> Full article ">Figure 2
<p>Influence of PJ-34 and DAPT inhibitors on proliferation and viability of leukemic cell lines. Jurkat, 697 and CLL cell lines were treated with PARP1 inhibitor PJ-34 (<b>A</b>) and Notch inhibitor DAPT (<b>B</b>) every second day, for six days. Total cell number and the fraction of living cells were obtained by counting cells stained with Trypan blue dye under the light microscope. The standard deviation of the four different counts is displayed. C: control cells; 10 µM, 20 µM, 40 µM, and 50 µM: concentrations of the treating agent; * <span class="html-italic">p</span>-value &lt; 0.05.</p> Full article ">Figure 3
<p>Influence of Notch and PARP pathways’ inhibition in Jurkat, CLL, and 697 cell lines. Cells were treated with PARP inhibitor PJ-34 and/or Notch inhibitor DAPT every other day through nine days, when RNA was isolated and, after reverse transcription, qRT-PCR was performed. Representative results are shown. Relative expression is presented as a fold change in comparison with untreated control sample values. As endogenous control gene <span class="html-italic">HPRT</span> expression was used. C: control cells; PJ-34: cells treated with PJ-34 (10 µM for CLL and Jurkat cells and 40 µM for 697); DAPT: 20 µM DAPT; PJ-34/DAPT: cells treated with combination of 10 µM PJ-34 and 20 µM DAPT; * <span class="html-italic">p</span>-value &lt; 0.05.</p> Full article ">Figure 4
<p>Influence of Notch and PARP pathways’ inhibition on protein expression. T cell line (Jurkat), B chronic lymphocytic cell line (CLL) and B cell precursor leukemia cell line (697) were treated with Notch pathway inhibitor DAPT (20 µM) or PARP inhibitor PJ-34 (10 µM) for nine days, when proteins were isolated and analyzed by Western blot. CTF: C-terminal fragment; ACTB: β-actin.</p> Full article ">
11 pages, 823 KiB  
Protocol
A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
by Johanna M. Buschhaus, Brock Humphries, Kathryn E. Luker and Gary D. Luker
Cells 2018, 7(6), 57; https://doi.org/10.3390/cells7060057 - 13 Jun 2018
Cited by 16 | Viewed by 5791
Abstract
Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic [...] Read more.
Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime. Cellular apoptosis was quantified in multiple environments ranging from 2D flat and 3D spheroid cell culture systems to in vivo murine mammary tumor xenografts. We evaluated cell-by-cell apoptotic responses to multiple pharmacological and genetic methods of interest involved in cancer cell death. Within this article, we describe methods for measuring caspase-3 activation at single-cell resolution in various complex environments using FLIM. Full article
(This article belongs to the Special Issue Innovative Methods to Monitor Single Live Cells)
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<p>Schematic representation of the LSS-mOrange-DEVD-mKate2 reporter. The intact DEVD linker between LSS-mOrange and mKate2 causes the two fluorescent proteins to be spatially close and experience increased FRET. This increase in FRET causes a decrease in the donor (LSS-mOrange) lifetime. Once the DEVD linker is enzymatically cleaved by active caspase-3, LSS-mOrange and mKate2 can spatially separate. This causes FRET to decrease and the donor (LSS-mOrange) lifetime to increase.</p> Full article ">Figure 2
<p>MDA-MB-231 cells show increased apoptosis with nutrient starvation. Left. MDA-MB-231 cells that have been starved of both glutamine and glucose (<b>bottom</b>) show a higher proportion of red pixels (indicating apoptosis) than the control, nutrient-rich condition (<b>top</b>). <b>Right</b>. The phasor plot indicates the ROIs that were selected. The numbers below the phasor plot indicate ROI spatial information. Points nearest the origin on the <span class="html-italic">x</span>-axis have a longer lifetime. The orange ROI indicates pixels with a shorter lifetime (no caspase-3 activity, FRET is occurring) and the red ROI indicates pixels with a longer lifetime (caspase-3 activity and apoptosis, no FRET).</p> Full article ">
24 pages, 2567 KiB  
Review
Unfolding the Endoplasmic Reticulum of a Social Amoeba: Dictyostelium discoideum as a New Model for the Study of Endoplasmic Reticulum Stress
by Eunice Domínguez-Martín, Mariana Hernández-Elvira, Olivier Vincent, Roberto Coria and Ricardo Escalante
Cells 2018, 7(6), 56; https://doi.org/10.3390/cells7060056 - 10 Jun 2018
Cited by 14 | Viewed by 8516
Abstract
The endoplasmic reticulum (ER) is a membranous network with an intricate dynamic architecture necessary for various essential cellular processes. Nearly one third of the proteins trafficking through the secretory pathway are folded and matured in the ER. Additionally, it acts as calcium storage, [...] Read more.
The endoplasmic reticulum (ER) is a membranous network with an intricate dynamic architecture necessary for various essential cellular processes. Nearly one third of the proteins trafficking through the secretory pathway are folded and matured in the ER. Additionally, it acts as calcium storage, and it is a main source for lipid biosynthesis. The ER is highly connected with other organelles through regions of membrane apposition that allow organelle remodeling, as well as lipid and calcium traffic. Cells are under constant changes due to metabolic requirements and environmental conditions that challenge the ER network’s maintenance. The unfolded protein response (UPR) is a signaling pathway that restores homeostasis of this intracellular compartment upon ER stress conditions by reducing the load of proteins, and by increasing the processes of protein folding and degradation. Significant progress on the study of the mechanisms that restore ER homeostasis was achieved using model organisms such as yeast, Arabidopsis, and mammalian cells. In this review, we address the current knowledge on ER architecture and ER stress response in Dictyostelium discoideum. This social amoeba alternates between unicellular and multicellular phases and is recognized as a valuable biomedical model organism and an alternative to yeast, particularly for the presence of traits conserved in animal cells that were lost in fungi. Full article
(This article belongs to the Special Issue Cellular Stress Response in Health and Disease)
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<p>Diagram of the <span class="html-italic">Dictyostelium</span> life cycle. Individual amoebas feed on yeast and bacteria, and multiply via fission. When nutrients are scarce, cells aggregate and undergo a developmental program, comprised of distinct stages that culminate in the formation of a fruiting body, which is composed of a stalk, and a sorogen filled with spores. Under suitable environmental conditions, the spores germinate.</p> Full article ">Figure 2
<p>The <span class="html-italic">Dictyostelium</span> endoplasmic reticulum (ER). In vivo confocal microscopy pictures showing a cortical section and a mid-section of a wild-type (WT) cell expressing the ER marker, Inositol requiring enzyme A (IreA) fused to the GFP. The asterisk pinpoints the nucleus, surrounded by the perinuclear ER. Arrows highlight zones where sheet-like regions are evident. Tubules can be distinguished across the entire cell area. (Scale bar represents 5 μm).</p> Full article ">Figure 3
<p>Signaling pathways involved in the unfolded protein response (UPR). In mammalian cells, three signaling branches that depend on the ER transmembrane sensor proteins—activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK), and inositol-requiring enzyme 1 (IRE1)—are activated upon ER stress (ERS). PERK and IRE1 can sense ERS by interacting directly with unfolded proteins through their luminal sensor domain. In addition, ATF6, PERK, and IRE1 detect an increase in unfolded proteins when they lose their association with the ER chaperone GRP78/binding immunoglobulin protein (BiP). When these transducers detect ERS, a recovery response is activated. This response mainly regulates two events: the reduction of ER protein load, and an increase in the protein-folding and degradation capacity of the cell. The former is accomplished via translation inhibition, triggered by the PERK-mediated phosphorylation of the eukaryotic initiation factor 2α (eIF2α), and by the degradation of certain messenger RNAs (mRNAs) in the regulated IRE1-dependent decay (RIDD). The second event regulates the activation or translation of transcription factors that, when transported to the nucleus, reprogram transcription to increase the expression of ER homeostatic genes, thus promoting protein folding and modification of the ER.</p> Full article ">Figure 4
<p>(<b>A</b>) Diagram of the structural domains of <span class="html-italic">Dictyostelium</span> IreA, compared with its <span class="html-italic">Saccharomyces cerevisiae</span> and human orthologs. SP (signal peptide), TM (transmembrane domain), KN (kinase domain), and KEN (kinase extension nuclease domain). Proteins were drawn to scale. Numbers indicate amino acid coordinates. Protein domains were obtained from <a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>. (<b>B</b>) Live-cell confocal microscopy of <span class="html-italic">ireA<sup>−</sup></span> cells expressing the IreA-GFP construct after 4 h, in the absence or in the presence of an ER-stress inducer. The IreA-GFP signal forms large puncta (possibly high-order oligomers). (Scale bar corresponds to 10 μm).</p> Full article ">Figure 5
<p>WT and <span class="html-italic">ireA<sup>−</sup></span> cells, after an ER stress treatment or mock, were fixed and prepared for the detection of the ER-resident protein disulfide isomerase (PDI) via an immunofluorescence assay and were visualized using confocal microscopy. An ER stress treatment severely impaired the ER morphology of the sensitive <span class="html-italic">ireA<sup>−</sup></span> cells. (Scale bar corresponds to 5 μm).</p> Full article ">Figure 6
<p>(<b>A</b>) Descriptive diagram of a serial dilution spotting assay used to test if a certain strain is sensitive to an ER stress inducer. A culture of bacterial cells (<span class="html-italic">Dictyostelium</span> is usually fed with <span class="html-italic">Klebsiella aerogenes</span> or <span class="html-italic">Escherichia coli</span>) is grown to saturation, and an aliquot is spread over an SM agar plate. Axenically growing <span class="html-italic">Dictyostelium</span> strains in the mid-logarithmic growth phase (with a density of around 1 × 10<sup>6</sup> cells/mL) are prepared and treated for the desired times with the ER stress inducer. After the treatment, <span class="html-italic">Dictyostelium</span> cells are collected, and serial dilutions are prepared and spotted on the SM agar plates. Plates are incubated at 22 °C until lysis plaques emerge due to the presence of growing amoebas feeding on bacteria. (<b>B</b>) Light microscopy pictures of WT and ER stress-sensitive <span class="html-italic">ireA<sup>−</sup></span> cells treated with a stress inducer. Morphological changes and cell lysis can be analyzed before the spotting assay. Notice the presence of round cells and the cell debris in the <span class="html-italic">ireA<sup>−</sup></span> strain after the treatment. (<b>C</b>) Picture of a spotting assay where a WT and an ER stress-sensitive strain (<span class="html-italic">ireA<sup>−</sup></span> cells) were tested with mock or ER stress inducer treatment.</p> Full article ">
21 pages, 3895 KiB  
Review
Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology
by Gonca Bulbul, Gepoliano Chaves, Joseph Olivier, Rifat Emrah Ozel and Nader Pourmand
Cells 2018, 7(6), 55; https://doi.org/10.3390/cells7060055 - 6 Jun 2018
Cited by 27 | Viewed by 8286
Abstract
Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding [...] Read more.
Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology. Full article
(This article belongs to the Special Issue Innovative Methods to Monitor Single Live Cells)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b>) Schematic representation of cell-surface detection by a double-barrel nanopipette; (<b>B</b>) SEM image shows the gold-sputtered double-barrel nanopipette; (<b>C</b>) Injection of carboxyfluorescein into human fibroblasts. The fluorescence intensity was normalized to that measured at 500 ms. Applied voltage: 10 V, scale bars 50 μm. The red curve is a sigmoidal fit to the experimental data points. (Reproduced from [<a href="#B27-cells-07-00055" class="html-bibr">27</a>] with the permission of the Royal Society of Chemistry).</p> Full article ">Figure 2
<p>(<b>A</b>) Fluorescence; (<b>B</b>) Bright-field merges show injections of green fluorescent protein (GFP), rhodamine, and mitotracker orange into the cells. GFP: green channel; mitotracker orange: blue channel; rhodamine: red channel. Cells stained purple are a mix of blue (mitotracker) and red (rhodamine) channels. One cell at center can be seen with GFP, mitotracker and rhodamine fluorescence, indicating three independent nanopipette interrogations. GFP was the first component to be injected into the cell, however it did not diffuse well into the cell, probably due to protein viscosity. After GFP, mitochondria-staining dye mitotracker orange was introduced. Rhodamine was injected as the third component into the group of cells. (Pourmand Lab, Personal Communication, 2018).</p> Full article ">Figure 3
<p>Aspiration of nuclear content by Nanopipette. (<b>A</b>) Nanopipette is placed on top of MCF-7 cell; (<b>B</b>) Nanopipette is placed on top of a different MCF-7 cell; (<b>C</b>) Fluorescence corresponding to mitotracker orange staining of cells depicted in (<b>A</b>); (<b>D</b>) Fluorescence corresponding to mitotracker orange staining of cells depicted in (<b>B</b>). Nuclear region is visualized by pattern of staining with the mitotracker dye. In (<b>D</b>) red arrow points dark compartment, corresponding to one nucleus. Green arrow shows one cytoplasmic region. Nanopipette was inserted into the nucleus, as seen in (<b>B</b>). Nuclear content was aspirated and transferred to the cDNA synthesis master mix, followed by sequencing using the Illumina Miseq. (Pourmand Lab, Personal Communication, 2018).</p> Full article ">Figure 4
<p>Limit of detection of ERCC. Content from the nanobiopsy of the nucleus was transferred to the cDNA mix (containing 0.5 µL of ERCC mixture at a 1:10,000 dilution) to reverse transcribe the RNAs followed by DNA sequencing. The sequencing reads were mapped to the ERCC reference pseudo-genome. The number of transcripts were counted using the HTSeq package and plotted as a function of the number of the ERCC transcripts (ERCC concentration × volume × dilution factor). The estimated intersect of the ERCC curve with the X axis was between 7 and 220, which represents at least one detected ERCC transcript. The threshold for detected transcripts was chosen to be 10 for subsequent analysis. (Pourmand Lab, Personal Communication, 2018).</p> Full article ">Figure 5
<p>Principal Component Analysis of gene expression in the nuclear nanobiopsy samples. (<b>A</b>) Raw data input to DESeq2; (<b>B</b>) DESeq2 run with log-normalized reads; (<b>C</b>) Resolution of clustering after removal of the MBL1, MBL9, MBL12 and MBL12 libraries as outliers; (<b>D</b>) Resolution of clustering excluding sequencing libraries MBL2 and MBL4. (Pourmand Lab, Personal Communication, 2018).</p> Full article ">Figure 6
<p>Principal Component Analysis of gene expression comparing two cell types at a time. (<b>A</b>–<b>C</b>) comparison of MDA-MB-231 and MCF-7 libraries cluster separately by cell type, seen as a trend in which same-cell type libraries cluster closer to each other; (<b>D</b>–<b>F</b>) comparison of HeLa vs. iCell Neurons cells. Libraries cluster separately by cell type. (Pourmand Lab, Personal Communication, 2018)</p> Full article ">Figure 7
<p>(<b>A</b>) Representative schematic showing the steps of glucose oxidase immobilization to the surface of the nanopipette tip. First, PLL is coated on the surface. Then, gluteraldehyde treatment occurs to cross-link the glucose oxidase to the PLL-coated surface; (<b>B</b>) After each step of immobilization, the changes were characterized electrochemically. 10 mM PBS (pH 7) was used as the supporting electrode; (<b>C</b>) Nanopipette tip imaged by SEM. Tip geometry is displayed in the inset; (<b>D</b>) Enzymatic process for conversion of glucose into hydrogen peroxide and gluconic acid. (Reprinted with the permission from [<a href="#B90-cells-07-00055" class="html-bibr">90</a>]. Copyright (2018) American Chemical Society).</p> Full article ">Figure 8
<p>Schematic representation of electrochemical configuration and pH monitoring in a single cell with a chitosan-modified nanopipette. (Reproduced from [<a href="#B96-cells-07-00055" class="html-bibr">96</a>] with the permission of the Royal Society of Chemistry).</p> Full article ">Figure 9
<p>Schematics showing the electrochemical configuration (top left) and the reversible binding of copper ions on the chitosan/PAA-modified nanopipette. (Reprinted with permission from [<a href="#B101-cells-07-00055" class="html-bibr">101</a>]. Copyright (2018) American Chemical Society).</p> Full article ">
26 pages, 597 KiB  
Article
Tensor Decomposition-Based Unsupervised Feature Extraction Can Identify the Universal Nature of Sequence-Nonspecific Off-Target Regulation of mRNA Mediated by MicroRNA Transfection
by Y.-H. Taguchi
Cells 2018, 7(6), 54; https://doi.org/10.3390/cells7060054 - 4 Jun 2018
Cited by 7 | Viewed by 4917
Abstract
MicroRNA (miRNA) transfection is known to degrade target mRNAs and to decrease mRNA expression. In contrast to the notion that most of the gene expression alterations caused by miRNA transfection involve downregulation, they often involve both up- and downregulation; this phenomenon is thought [...] Read more.
MicroRNA (miRNA) transfection is known to degrade target mRNAs and to decrease mRNA expression. In contrast to the notion that most of the gene expression alterations caused by miRNA transfection involve downregulation, they often involve both up- and downregulation; this phenomenon is thought to be, at least partially, mediated by sequence-nonspecific off-target effects. In this study, I used tensor decomposition-based unsupervised feature extraction to identify genes whose expression is likely to be altered by miRNA transfection. These gene sets turned out to largely overlap with one another regardless of the type of miRNA or cell lines used in the experiments. These gene sets also overlap with the gene set associated with altered expression induced by a Dicer knockout. This result suggests that the off-target effect is at least as important as the canonical function of miRNAs that suppress translation. The off-target effect is also suggested to consist of competition for the protein machinery between transfected miRNAs and miRNAs in the cell. Because the identified genes are enriched in various biological terms, these genes are likely to play critical roles in diverse biological processes. Full article
(This article belongs to the Special Issue Regulatory microRNA)
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<p>The results on the artificial data: (<b>A</b>) <math display="inline"><semantics> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>2</mn> </msub> <mo>=</mo> <mn>1</mn> <mo>,</mo> <mi>j</mi> </mrow> </msub> </semantics></math> averaged across 100 independent trials. The horizontal red dashed line is <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>2</mn> </msub> <mo>=</mo> <mn>1</mn> <mo>,</mo> <mi>j</mi> </mrow> </msub> <mo>=</mo> <mn>0</mn> </mrow> </semantics></math> (<b>B</b>) <math display="inline"><semantics> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>3</mn> </msub> <mo>=</mo> <mn>1</mn> <mo>,</mo> <mi>k</mi> </mrow> </msub> </semantics></math> averaged across 100 independent trials. The horizontal red dashed line is <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>3</mn> </msub> <mo>=</mo> <mn>1</mn> <mo>,</mo> <mi>k</mi> </mrow> </msub> <mo>=</mo> <mn>0</mn> </mrow> </semantics></math> (<b>C</b>) A histogram of <math display="inline"><semantics> <mrow> <mn>1</mn> <mo>−</mo> <mi>P</mi> </mrow> </semantics></math> computed from <math display="inline"><semantics> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>1</mn> <mo>,</mo> <mi>i</mi> </mrow> </msub> </semantics></math>. A vertical red segment represents the bin with the smallest <span class="html-italic">P</span>-values.</p> Full article ">Figure 2
<p>A schematic diagram that summarizes the obtained results.</p> Full article ">Figure 3
<p>A boxplot of the number of miRNAs that target individual genes as a function of the number of experiments that select individual genes within 11 experiments (most frequently selected genes were selected in nine experiments): (<b>Left</b>) raw numbers (Pearson’s correlation coefficient = 0.13, <math display="inline"><semantics> <mrow> <mi>P</mi> <mo>=</mo> <mn>3.9</mn> <mo>×</mo> <msup> <mn>10</mn> <mrow> <mo>−</mo> <mn>11</mn> </mrow> </msup> </mrow> </semantics></math>); and (<b>Right</b>) ranks of numbers (Spearman’s correlation coefficient = 0.29, <math display="inline"><semantics> <mrow> <mi>P</mi> <mo>&lt;</mo> <mn>2.2</mn> <mo>×</mo> <msup> <mn>10</mn> <mrow> <mo>−</mo> <mn>16</mn> </mrow> </msup> </mrow> </semantics></math>)</p> Full article ">Figure 4
<p>(<b>A</b>) The scatter plot of <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>1</mn> </msub> <mo>,</mo> <mi>i</mi> </mrow> </msub> <mo>,</mo> <msub> <mi>ℓ</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>1</mn> <mrow> <mo stretchy="false">(</mo> <mrow> <mi>horizontal</mi> <mi>axis</mi> </mrow> <mo stretchy="false">)</mo> </mrow> <mo>,</mo> <mn>2</mn> <mrow> <mo stretchy="false">(</mo> <mrow> <mi>vertical</mi> <mi>axis</mi> </mrow> <mo stretchy="false">)</mo> </mrow> </mrow> </semantics></math> for GSE26996; 379 red dots are selected probes; and (<b>B</b>) a semilogarithmic plot of the histogram of <math display="inline"><semantics> <mrow> <mn>1</mn> <mo>−</mo> <mi>P</mi> </mrow> </semantics></math> under the null hypothesis that <math display="inline"><semantics> <msub> <mi>x</mi> <mrow> <msub> <mi>ℓ</mi> <mn>1</mn> </msub> <mo>=</mo> <mn>2</mn> <mo>,</mo> <mi>i</mi> </mrow> </msub> </semantics></math> obeys a normal distribution. A sharp peak is observed in the red bin with the largest <math display="inline"><semantics> <mrow> <mn>1</mn> <mo>−</mo> <mi>P</mi> </mrow> </semantics></math>, which includes all the probes selected in (<b>A</b>).</p> Full article ">
29 pages, 3691 KiB  
Review
The Nutrient-Sensing Hexosamine Biosynthetic Pathway as the Hub of Cancer Metabolic Rewiring
by Ferdinando Chiaradonna, Francesca Ricciardiello and Roberta Palorini
Cells 2018, 7(6), 53; https://doi.org/10.3390/cells7060053 - 2 Jun 2018
Cited by 103 | Viewed by 18282
Abstract
Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). [...] Read more.
Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the “sensing molecule” UDP-N-Acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is the substrate for the enzymes involved in protein N- and O-glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O- and N-glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways. Full article
(This article belongs to the Section Mitochondria)
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<p>Schematic representation of the hexosamine biosynthesis pathway. HBP enzymes are depicted in red, metabolites in black, cellular processes in light blue. The green/red boxes indicate the metabolites whose change induces UDP-Glc<span class="html-italic">N</span>Ac decrease (green) or increase (red).</p> Full article ">Figure 2
<p>Schematic representation of the nutrient transporters discussed in the text. The arrows in the transporters indicate the direction of the flux. The red arrows indicate the increased nutrient transport and/or transporter affinity/targeting/stability. The single or double symbol of <span class="html-italic">N</span>-glycosylation indicates low or high glycosylated protein, respectively. The cartoon representing endoplasmic reticulum and Golgi indicates stability and targeting. Gluc: glucose; Lac: lactate; Gln: glutamine; AA: amino acid; <span class="html-small-caps">l</span>-Car: <span class="html-small-caps">l</span>-carnitine.</p> Full article ">Figure 3
<p>HBP modulates cancer metabolism through the <span class="html-italic">O</span>-Glc<span class="html-italic">N</span>Acylation of metabolic enzymes, signaling proteins and mitochondrial proteins. (<b>A</b>) In the image, the main metabolic pathways exploited by cancer cells for anabolism and bioenergetics are represented. The <span class="html-italic">O</span>-Glc<span class="html-italic">N</span>Acylated proteins (mainly the metabolic enzymes) are indicated in the grey boxes if the effect of the glycosylation has been demonstrated. The circle on the box, representing the <span class="html-italic">O</span>-Glc<span class="html-italic">N</span>Acylation, is red when the protein function is activated by glycosylation and green when it is inhibited. (<b>B</b>) The reciprocal regulation of AMPK and PKA and the HBP is schematically represented as discussed in the main text. 6-PG, 6-phosphogluconate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; THF, tetrahydrofolate; DHF, dihydrofolate; FA, fatty acids; Fatty Ac-CoA, Fatty Acyl-Coenzyme A; GSH, glutathione; Trx, thioredoxin; NT, nucleotides.</p> Full article ">Figure 4
<p>Schematic representation of the main metabolic pathways influenced (positively and/or negatively) by transcriptional factor <span class="html-italic">O-</span>Glc<span class="html-italic">N</span>Acylation. The figure depicts, from a schematic point of view, the transcriptional factors whose stability, transcriptional activation, DNA binding ability and protein–protein interaction is regulated by direct <span class="html-italic">O-</span>Glc<span class="html-italic">N</span>Acylation and the metabolic pathways influenced by their <span class="html-italic">O-</span>Glc<span class="html-italic">N</span>Acylation. The colored square around the indicated metabolic pathways is related to the specific transcriptional factor that upon <span class="html-italic">O-</span>Glc<span class="html-italic">N</span>Acylation is able to control the pathway.</p> Full article ">
20 pages, 1046 KiB  
Review
Remarkable Progress with Small-Molecule Modulation of TRPC1/4/5 Channels: Implications for Understanding the Channels in Health and Disease
by Aisling Minard, Claudia C. Bauer, David J. Wright, Hussein N. Rubaiy, Katsuhiko Muraki, David J. Beech and Robin S. Bon
Cells 2018, 7(6), 52; https://doi.org/10.3390/cells7060052 - 1 Jun 2018
Cited by 43 | Viewed by 7176
Abstract
Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na+, K+ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for [...] Read more.
Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na+, K+ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes. Full article
(This article belongs to the Special Issue TRP Channels in Health and Disease)
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Graphical abstract
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<p>(<b>A</b>) Formation of tetrameric TRPC1/4/5 cation channels by TRPC1/4/5 proteins and recently discovered small-molecule modulators discussed in this review. Domains “A” are the ankyrin repeat domains present in all TRPC proteins, and “P” is the PDZ binding domain present in TRPC4 and TRPC5. (<b>B</b>) Composition of different TRPC1/4/5 channels discussed in this review. Native channels are believed to exist predominantly as heteromers, but their exact compositions and stoichiometries are often unknown (as depicted by white subunits, which may be TRPC1/4/5 proteins or other TRP(C) proteins). Linked subunits depict recombinant TRPC4–C1 and TRPC5–C1 concatemeric proteins that can be used to control channel stoichiometry.</p> Full article ">Figure 2
<p>Example I–V plots of homomeric (<b>left</b>) and heteromeric/concatemeric (<b>right</b>) TRPC1/4/5 channels. Recordings from overexpressed TRPC4:C4 and TRPC1:C4 channels are shown. For examples of native I–V plots, see references [<a href="#B14-cells-07-00052" class="html-bibr">14</a>,<a href="#B16-cells-07-00052" class="html-bibr">16</a>].</p> Full article ">Figure 3
<p>Structures of recently reported TRPC1/4/5 activators, the (−)EA metabolite (−)EB, and the (−)EA antagonist A54.</p> Full article ">Figure 4
<p>Structures of ML204 and recently reported TRPC1/4/5 inhibitors.</p> Full article ">
28 pages, 3182 KiB  
Review
Fluorescent, Bioluminescent, and Optogenetic Approaches to Study Excitable Physiology in the Single Cardiomyocyte
by Connor N. Broyles, Paul Robinson and Matthew J. Daniels
Cells 2018, 7(6), 51; https://doi.org/10.3390/cells7060051 - 31 May 2018
Cited by 32 | Viewed by 10398
Abstract
This review briefly summarizes the single cell application of classical chemical dyes used to visualize cardiomyocyte physiology and their undesirable toxicities which have the potential to confound experimental observations. We will discuss, in detail, the more recent iterative development of fluorescent and bioluminescent [...] Read more.
This review briefly summarizes the single cell application of classical chemical dyes used to visualize cardiomyocyte physiology and their undesirable toxicities which have the potential to confound experimental observations. We will discuss, in detail, the more recent iterative development of fluorescent and bioluminescent protein-based indicators and their emerging application to cardiomyocytes. We will discuss the integration of optical control strategies (optogenetics) to augment the standard imaging approach. This will be done in the context of potential applications, and barriers, of these technologies to disease modelling, drug toxicity, and drug discovery efforts at the single-cell scale. Full article
(This article belongs to the Special Issue Innovative Methods to Monitor Single Live Cells)
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<p>Annual publications imaging cardiomyocytes or heart tissue using the fluorescent Ca<sup>2+</sup> indicators fura-2, fluo-4, and Rhod-2. Metrics extracted from CSV data at Pubmed.</p> Full article ">Figure 2
<p>Summary of the composition and mode of action of the genetically-encoded fluorescent sensors for Ca<sup>2+</sup>, voltage, and ATP discussed in the article.</p> Full article ">Figure 3
<p>A summary of optogenetic actuators. Depolarizing channelrhodopsins, e.g., ChR2, open in response to light in the blue-green spectrum allowing positively-charged ions into the cell, raising the membrane potential and triggering the depolarization threshold. Conversely, inhibitory channels like Halorhodopsin (which pumps negatively-charged ions in) or Archaerhodopsin (which pumps protons out) hyperpolarize excitable membranes. Inhibitory channel activity is controlled by light in the green-red spectrum. The lower panel stylizes the effect on cell membrane potential by blue or orange pulses of light to depolarize, or hyperpolarize the cell. The two approaches can be combined to hyperpolarize, and then activate, the cell [<a href="#B95-cells-07-00051" class="html-bibr">95</a>]. A number of reporter strategies discussed in this review can be integrated with stimulatory optical control [<a href="#B42-cells-07-00051" class="html-bibr">42</a>,<a href="#B60-cells-07-00051" class="html-bibr">60</a>,<a href="#B68-cells-07-00051" class="html-bibr">68</a>,<a href="#B69-cells-07-00051" class="html-bibr">69</a>,<a href="#B78-cells-07-00051" class="html-bibr">78</a>,<a href="#B81-cells-07-00051" class="html-bibr">81</a>,<a href="#B83-cells-07-00051" class="html-bibr">83</a>,<a href="#B85-cells-07-00051" class="html-bibr">85</a>,<a href="#B97-cells-07-00051" class="html-bibr">97</a>].</p> Full article ">
14 pages, 2136 KiB  
Article
B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody
by Philipp Fecher, Richard Caspell, Villian Naeem, Alexey Y. Karulin, Stefanie Kuerten and Paul V. Lehmann
Cells 2018, 7(6), 50; https://doi.org/10.3390/cells7060050 - 31 May 2018
Cited by 15 | Viewed by 6660
Abstract
In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also [...] Read more.
In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to—and largely limited by—the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC. Full article
(This article belongs to the Special Issue Recent Advances in ELISPOT Research)
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<p>Unambiguous detection of the four fluorescent tags identifying the individual IgG subclasses. In each row, a single-color B cell ELISPOT assay was performed using the respective IgG subclass-specific detection reagent, as specified in the figure. Each assay was analyzed with filter settings optimized for the individual colors. Across three independent experiments (of which representative scans are shown), individual florescent channels (FL1 to FL4), readily detected corresponding fluorescent tags. Importantly, no cross-bleeding of colors between channels occurred.</p> Full article ">Figure 2
<p>Representative four-color well. The membrane was coated with anti-κ/λ capture antibody and polyclonally activated peripheral blood mononuclear cells (PBMC) were plated at 50,000 cells/well. Detection regents for each of the four IgG subclasses were added to visualize the membrane-bound IgG molecules. Images were captured for each individual fluorescent channel (see <a href="#cells-07-00050-f001" class="html-fig">Figure 1</a>) and superimposed using the following artificially assigned colors for each color plane: IgG1: Red; IgG2: Yellow; IgG3: Blue; and IgG4: Green.</p> Full article ">Figure 3
<p>Reproducibility of the four color B cell assay. To assess inter-assay variation in spot numbers, PBMC were stimulated polyclonally, and frozen in aliquots on day four. Three independent assays were performed after thawing an aliquot, measuring the four IgG subclasses on anti-κ/λ capture antibody-coated membranes. Each bar represents the mean spot count + SD for the tests done in four replicate wells.</p> Full article ">Figure 4
<p>Calculating frequencies for pan IgG-SFU/million PBMC illustrated. Polyclonally stimulated PBMC were plated in the specified cell numbers in quadruplicate wells, and a Three Color ImmunoSpot<sup>®</sup> assay was performed for Ig classes pan-IgG, M and A. (<b>A</b>) Raw images for the IgG color plane are shown with the cell numbers plated per well specified; (<b>B</b>) the mean spot count for each cell concentration, with the SD for the quadruplicate wells tested is plotted vs. the cell numbers plated. As the spots at high ASC densities start crowding (plus when the number of ASC is high, there is an “ELISA” effect with analyte captured from the supernatant causing a high background carpet staining), for high cell numbers Spot Forming Units (SFU) become no longer precisely countable: “too numerous to count is shown by a star; (<b>C</b>) While at high numbers spot counts tend to deviate from linearity due to crowding, in lower numbers they followed a liner relationship between cell numbers plated and SFU counted, in this case with an <span class="html-italic">R</span><sup>2</sup> value of 0.9939. Based on the regression line shown in red, the pan-IgG SFU frequency has been calculated to be, in the example shown here, 13,800 pan-IgG SFU per one million PBMC.</p> Full article ">Figure 5
<p>Effect of cryopreservation on Ig class production by B cells. Freshly isolated PBMC were polyclonally stimulated for four days and then tested in a three color B cell ImmunoSpot<sup>®</sup> assay, with serial dilution of the cells in four replicate wells, (“Fresh”), as illustrated in <a href="#cells-07-00050-f004" class="html-fig">Figure 4</a>. The same PBMC were cryopreserved, thawed, and then polyclonally stimulated, seeded and tested as above (“Frozen”). The latter cells were cryopreserved at the end of the four day polyclonal stimulation culture, when B cell blasts have been engaged, then thawed, and seeded and tested as above (“Blasts”). The relationship of Fresh/Frozen and Blast cells is graphically illustrated in <a href="#app1-cells-07-00050" class="html-app">Figure S1</a>. The results obtained testing 15 PBMC donors are shown here for pan-IgG (<b>A</b>), IgM (<b>B</b>), and IgA (<b>C</b>). The SFU counts per million PBMC have been established as specified in <a href="#cells-07-00050-f004" class="html-fig">Figure 4</a>. For each donor, as defined by color in the insert, the mean spot counts are connected by the corresponding color coded lines.</p> Full article ">Figure 6
<p>Effect of cryopreservation on IgG subclass production by B cells. The legend to <a href="#cells-07-00050-f005" class="html-fig">Figure 5</a> applies except that a four color B cell ImmunoSpot<sup>®</sup> assay was performed measuring IgG1 (<b>A</b>), IgG2 (<b>B</b>), IgG3 (<b>C</b>), and IgG4 (<b>D</b>) on the serially diluted PBMC. Results obtained for 15 PBMC donors tested are shown.</p> Full article ">
8 pages, 1126 KiB  
Article
Novel Modulators of Proteostasis: RNAi Screen of Chromosome I in a Heat Stress Paradigm in C. elegans
by Andreas Kern, Natalie Spang, Heike Huesmann and Christian Behl
Cells 2018, 7(6), 49; https://doi.org/10.3390/cells7060049 - 26 May 2018
Cited by 6 | Viewed by 4747
Abstract
Proteostasis is of vital importance for cellular function and it is challenged upon exposure to acute or chronic insults during neurodegeneration and aging. The proteostasis network is relevant for the maintenance of proteome integrity and mainly comprises molecular chaperones and two degradation pathways, [...] Read more.
Proteostasis is of vital importance for cellular function and it is challenged upon exposure to acute or chronic insults during neurodegeneration and aging. The proteostasis network is relevant for the maintenance of proteome integrity and mainly comprises molecular chaperones and two degradation pathways, namely, autophagy and the ubiquitin proteasome system. This network is characterized by an impressive functional interrelation and complexity, and occasionally novel factors are discovered that modulate proteostasis. Here, we present an RNAi screen in C. elegans, which aimed to identify modulators of proteostasis in a heat stress paradigm. The screen comprised genes that are located on chromosome I of the nematode and has identified 185 genetic modifiers, whose knockdown has enhanced the misfolding of a reporter protein upon temperature increase. Subsequently, we evaluated the effect of a distinct number of the identified candidates in an additional C. elegans model strain, which expresses the aggregation-prone PolyQ35::YFP protein. Moreover, we annotated the human orthologs of the identified proteins and analyzed their enrichment in functional clusters and, as appropriate, their association with human neuropathologies. The achieved data collection includes several factors that have already been functionally associated with the proteostasis network, which highlights the potential of this heat stress-based proteostasis screen in order to detect novel modulators of proteome integrity. Full article
(This article belongs to the Special Issue Autophagy in Age-Related Human Diseases)
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<p>Evaluation of the heat stress-based RNAi screen. (<b>A</b>) Schematic sequence of the RNAi approach and representative fluorescence images of body wall muscle cells of LUC::GFP-expressing worms after heat stress and the respective RNAi treatment. Scale bar: 20 µm. (<b>B</b>) Statistics of first and second trial. Initially, 218 candidates were identified, of which 85 % repeated their effects on LUC::GFP upon heat stress. (<b>C</b>) Representative fluorescence images of PolyQ35::YFP after RNAi treatment. Arrows indicate aggregates. Scale bar: 100 µm. The table summarizes candidates that were evaluated for their impact on PolyQ35::YFP aggregation. Green square: increased aggregation; Red square: no impact on aggregation. (<b>D</b>) Human orthologs of identified candidates were analyzed for functional clusters employing the bioinformatics tool DAVID. The tables depict candidates functionally associated with the ubiquitin conjugation pathway or autophagy, respectively.</p> Full article ">Figure 2
<p>Association of identified candidates with human neuropathologies. Human orthologs were linked to neuropathologies employing online resources and are represented relative to their total number. The table lists identified candidates related to Alzheimer disease</p> Full article ">
31 pages, 1295 KiB  
Review
Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications
by Angela Di Baldassarre, Elisa Cimetta, Sveva Bollini, Giulia Gaggi and Barbara Ghinassi
Cells 2018, 7(6), 48; https://doi.org/10.3390/cells7060048 - 25 May 2018
Cited by 50 | Viewed by 7662
Abstract
Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of [...] Read more.
Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications. Full article
(This article belongs to the Special Issue Ten Years of iPSCs: Current Status and Future Perspectives)
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<p>(<b>A</b>). Schematic of the device: the central cell loading channel is connected to the lateral C-shaped medium-delivering channels by a “ladder” of thin microchannels, purposely design to protect cells from shear and switch to a diffusive mass transport regime. The colors are representative of the linear velocities on a chosen plane, and show decreasing values from red to blue; (<b>B</b>). Characterization of the 3D cardiac tissue formed in the middle channel. Top: optical microscopy image showing tissue density and overall organization and alignment; middle: heat map of the average motion generated by the contractile activity and, bottom: corresponding average beating kinetics. Adapted with permission from Mathur et al. [<a href="#B127-cells-07-00048" class="html-bibr">127</a>]; (<b>C</b>). Top: schematic representation of contracting constructs and approach to measurements; Bottom: iPSC-CMs seeded onto thin elastomers with patterned lines of fibronectin self-organized into microscaled myocardial tissues and exhibited contractile properties in response to electrical stimulation; (<b>D</b>). Representative images showing actinin staining of iPSC-CMs on micropatterned fibronectin rectangles. BTHS iPSC-CM micro-tissues show impaired sarcomere organization (BTHS1 and 2 in Galactose and Glucose medium, respectively), while cells transfected with TAZ modRNA (Mod BHTS) clearly demonstrate a rescued organization, comparable to that of the control cultures (CTRL). Adapted from Wang et al. [<a href="#B128-cells-07-00048" class="html-bibr">128</a>].</p> Full article ">
12 pages, 940 KiB  
Review
Experimental Methods for Studying Cellular Heme Signaling
by Jonathan M. Comer and Li Zhang
Cells 2018, 7(6), 47; https://doi.org/10.3390/cells7060047 - 24 May 2018
Cited by 12 | Viewed by 6055
Abstract
The study of heme is important to our understanding of cellular bioenergetics, especially in cancer cells. The function of heme as a prosthetic group in proteins such as cytochromes is now well-documented. Less is known, however, about its role as a regulator of [...] Read more.
The study of heme is important to our understanding of cellular bioenergetics, especially in cancer cells. The function of heme as a prosthetic group in proteins such as cytochromes is now well-documented. Less is known, however, about its role as a regulator of metabolic and energetic pathways. This is due in part to some inherent difficulties in studying heme. Due to its slightly amphiphilic nature, heme is a “sticky” molecule which can easily bind non-specifically to proteins. In addition, heme tends to dimerize, oxidize, and aggregate in purely aqueous solutions; therefore, there are constraints on buffer composition and concentrations. Despite these difficulties, our knowledge of heme’s regulatory role continues to grow. This review sums up the latest methods used to study reversible heme binding. Heme-regulated proteins will also be reviewed, as well as a system for imaging the cellular localization of heme. Full article
(This article belongs to the Section Mitochondria)
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<p>Hap1 repression modules (RPMs) promote Hap1 association with Ydj1, Ssa (Hsp70), and Sro9 to form an inactive High Molecular Weight Complex (HMC). Upon heme binding to Heme Responsive Motif 7 (HRM7), Hsp90 is stably bound and the HMC is disrupted, producing the active dimeric complex (DC). In the new complex, the Hap1 acidic activation domains (ACT) are activated and DNA binding is promoted via the DNA binding domains (DBD).</p> Full article ">Figure 2
<p>At low heme levels, the ZnF region and other proteins (X) repress Gis1 transcriptional and demethylase activities. At higher heme concentrations, heme binds a second site in the ZnF causing loss of X, oligomerization of Gis1, and conformational changes which fully activate Gis1 demethylase and transcriptional activities. JmjN and JmjC: jumonji domains and demethylase activity. TAD1 and TAD2: transcription activation domains. ZnF: C2H2 type zinc fingers.</p> Full article ">
13 pages, 10763 KiB  
Review
Mitochondrial Fatty Acid Oxidation Disorders Associated with Short-Chain Enoyl-CoA Hydratase (ECHS1) Deficiency
by Alice J. Sharpe and Matthew McKenzie
Cells 2018, 7(6), 46; https://doi.org/10.3390/cells7060046 - 23 May 2018
Cited by 60 | Viewed by 11522
Abstract
Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for [...] Read more.
Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for many organs and tissues, including the heart, liver and brain. Deficiencies in FAO can cause life-threatening metabolic disorders in early childhood that present with liver dysfunction, hypoglycemia, dilated hypertrophic cardiomyopathy and Reye-like Syndrome. Alternatively, FAO defects can also cause ‘milder’ adult-onset disease with exercise-induced myopathy and rhabdomyolysis. Short-chain enoyl-CoA hydratase (ECHS1) is a key FAO enzyme involved in the metabolism of fatty acyl-CoA esters. ECHS1 deficiency (ECHS1D) also causes human disease; however, the clinical manifestation is unlike most other FAO disorders. ECHS1D patients commonly present with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy traditionally associated with defects in oxidative phosphorylation (OXPHOS). In this article, we review the clinical, biochemical and genetic features of the ESHS1D patients described to date, and discuss the significance of the secondary OXPHOS defects associated with ECHS1D and their contribution to overall disease pathogenesis. Full article
(This article belongs to the Special Issue Mitochondrial Biology in Health and Disease)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Mitochondrial fatty acid β-oxidation (FAO). Enzymes of the carnitine shuttle system (yellow) are responsible for transporting fatty acyl-CoA esters into the mitochondrial matrix as acylcarnitines. Carnitine is added to fatty acyl-CoAs by carnitine <span class="html-italic">O</span>-palmitoyltransferase 1 (CPT1), forming acylcarnitines that are transported into the mitochondrial matrix by the carnitine acylcarnitine translocase (CACT). Once inside the mitochondrial matrix, carnitine <span class="html-italic">O</span>-palmitoyltransferase 2 (CPT2) removes the carnitine to regenerate the fatty acyl-CoA ester. Four reactions (<b>1</b>–<b>4</b>) then occur for each round of FAO, catalyzed by enzymes with different carbon chain length specificities (as shown): <b>1</b>—dehydrogenation of fatty acyl-CoA esters by very long-chain (VLCAD), medium-chain (MCAD), and short-chain (SCAD) acyl-CoA dehydrogenases (shown in green) to form enoyl-CoA, <b>2</b>—hydration of enoyl-CoA by the mitochondrial trifunctional protein (MTP, blue) or short-chain enoyl-CoA hydratase (ECHS1, red) to form 3-hydroxyacyl-CoA, <b>3</b>—dehydrogenation of 3-hydroxyacyl-CoA by MTP or hydroxyacyl-CoA dehydrogenase (HADH, purple) to form 3-ketoacyl-CoA, <b>4</b>—thiolysis of 3-ketoacyl-CoA by MTP or 3-ketoacyl-CoA thiolase (KAT, pink). The resulting fatty acyl-CoA is shortened by two carbons, with the generation of acetyl-CoA, NADH and FADH<sub>2</sub>. NADH and FADH<sub>2</sub> provide electrons for OXPHOS, while acetyl-CoA enters the TCA cycle to generate further NADH and FADH<sub>2</sub>. The shortened fatty acyl-CoA undergoes further rounds of FAO until only two acetyl-CoA molecules remain. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane.</p> Full article ">Figure 2
<p>ECHS1 Structure and Function. (<b>A</b>) Homohexameric ECHS1 crystal structure at 2.55 Å resolution (PDB: 2hw5), showing six ECHS1 units colored by chain. Two copies of the 4-carbon substrate crotonyl-CoA are shown (bottom right hand corner). (<b>B</b>) ECHS1 catalyzes the conversion of trans-Δ<sup>2</sup>-enoyl-CoA thioesters to 3-<span class="html-small-caps">l</span>-hydroxyacyl-CoA thioesters by stereospecific hydration of the trans double bond between carbons two and three. Hydration of crotonyl-CoA to 3-hydroxybutyryl-CoA is shown.</p> Full article ">
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