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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Different SUMO Paralogs Determine the Fate of WT and Mutant CFTRs: Biogenesis vs. Degradation

A pathway for CFTR degradation is initiated by Hsp27 which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogs form poly-chains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, PIAS4, which increased WT and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR three-fold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation 3-fold and stabilized mature WT CFTR at the PM. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralog specific effects of PIAS4 were reproduced in vitro using purified F508del NBD1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ~50%, and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogs determine the fates of WT and mutant CFTRs, and they suggest that a paralog switch during biogenesis can direct these proteins to different outcomes: biogenesis vs. degradation.

cell biology↗

bioRxiv2018

Adaptation of actin dynamics and membrane tension control for yeast endocytosis

During clathrin-mediated endocytosis in eukaryotes, actin assembly is required to overcome large membrane tension and turgor pressure. However, the molecular mechanisms by which the actin machinery adapts to varying membrane tension remain unknown. In addition, how cells reduce their membrane tension when they are challenged by hypotonic shocks remains unclear. We used quantitative microscopy to demonstrate that cells rapidly reduce their membrane tension using three parallel mechanisms. In addition to using their cell wall for mechanical protection, yeast cells disassemble eisosomes to buffer moderate changes in membrane tension on a minute time scale. Meanwhile, a temporary reduction of the rate of endocytosis for 2 to 6 minutes, and an increase in the rate of exocytosis for at least 5 minutes allow cells to add large pools of membrane to the plasma membrane. We built on these results to submit the cells to abrupt increases in membrane tension and determine that the endocytic actin machinery of fission yeast cells rapidly adapts to perform clathrin-mediated endocytosis. Our study sheds light on the tight connection between membrane tension regulation, endocytosis and exocytosis.

cell biology↗

bioRxiv2018

miR17~92 is essential for the survival of hematopoietic stem and progenitor cells by restraining pro-apoptotic BIM

The micro RNA cluster miR17~92, also known as oncomiR-1, impacts diverse cellular processes, such as cell survival and proliferation. Constitutive loss of miR17~92 in mice causes severe defects in skeletal development, organ development and hematopoiesis, resulting in early post-natal lethality. The critical functions of miR17~92 in a fully developed animal have not yet been explored. Here we show that deletion of miR17~92 in adult mice [Formula] had no impact on their lifespan or general well-being. However, detailed analysis of the hematopoietic system in [Formula] mice, revealed a dramatic reduction in all mature hematopoietic lineages, which was due to the loss of early hematopoietic stem/progenitor cells (HSPCs). Strikingly, the concomitant loss of the pro-apoptotic BH3-only protein BIM rescued the loss of the HSPCs and all of their differentiated progeny that was caused by the deletion of miR17~92. These findings demonstrate that miR17~92 is critical for the survival of HSPCs by restraining the activity of the pro-apoptotic BH3-only protein BIM.

cell biology↗

bioRxiv2018

Nrf1D is the first candidate secretory transcription factor in the blood plasma, with its precursor existing as a unique redox-sensitive transmembrane CNC-bZIP protein in somatic tissues

Amongst multiple distinct isoforms, Nrf1D is synthesized from translation of an alternatively-spliced transcript of Nrf1 mRNA, with a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1s C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was identified to fold into a redox-sensitive transmembrane domain that enables it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D confers it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with only a less ability than wild-type Nrf1 at mediating target gene expression. Further evidence has been presented revealing that both mRNA and protein levels of Nrf1D were detected to varying extents in somatic tissues. Surprisingly, we also found the existence of Nrf1D-derived isoforms in the blood plasma, implying that it is a candidate secretory transcription factor, although its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies, before being unleashed from the ER to enter the blood plasma.

cell biology↗

bioRxiv2018

Mapping of TNTs using Correlative Cryo-Electron Microscopy Reveals a Novel Structure

The harmonious orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions, called tunneling nanotubes, that allow for the intercellular transport of various cargoes, including viruses, organelles, and proteins between the cytoplasm of distant cells in vitro and in vivo. Over the last decade, studies have focused on their functional role but information regarding their structure and the differences with other cellular protrusions such as filopodia, is still lacking. Here, we report the structural characterization of tunneling nanotubes using correlative light- and cryo-electron microscopy approaches. We demonstrate their structural identity compared to filopodia by showing that they are comprised of a bundle of functional individual Tunneling Nanotubes containing membrane-bound compartments and allowing organelle transfer.

cell biology↗

bioRxiv2018

Regulation of FtsZ levels in Escherichia coli in slow growth conditions

A key regulator of cell division in most walled bacteria is the FtsZ protein that assembles into protofilaments attached to the membrane at midcell. These dynamic protofilament assemblies, known as the Z-ring, act as a scaffold for more than two dozen proteins involved in synthesis of septal cell envelopes. What triggers the formation of the Z-ring during the cell cycle is poorly understood. In Escherichia coli model organism, the common view is that FtsZ concentration is constant throughout its doubling time and therefore regulation of assembly should be controlled by some yet to be identified protein-protein interactions. Here we show using quantitative analysis of newly developed fluorescent reporter that FtsZ concentration varies in a cell-cycle dependent manner in slow growth conditions and that upregulation of FtsZ synthesis correlates with the formation of the Z-ring. About 4-fold upregulation of FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the last 10% of the cell cycle. The initiation of rapid degradation coincides with dissociation of FtsZ from the septum. Altogether, our data indicate that the Z-ring formation in slow growth conditions in E. coli is controlled by a regulatory sequence where upregulation of an essential cell cycle factor is followed by its degradation.\n\nSignificanceFtsZ is the key regulator for bacterial cell division. It initiates division by forming a dynamic ring-like structure, the Z-ring, at the mid-cell. Here we show that, contrarily to the current paradigm, FtsZ concentration in Escherichia coli model organism varies throughout cell cycle in slow growth conditions. Faster FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the end of the cell cycle. Upregulation of FtsZ synthesis correlates with the formation of the Z-ring. Our data demonstrates that in slow growth E. coli cell division progresses according to paradigm where upregulation of essential cell cycle factor is followed by its degradation.

cell biology↗

bioRxiv2018

Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.

cell biology↗

bioRxiv2018

Kinesin Binding Protein (KBP) buffers the activity of Kif18A and Kif15 in mitosis to ensure accurate chromosome segregation

Mitotic kinesins must be regulated to ensure a precise balance of spindle forces and accurate segregation of chromosomes into daughter cells. Here we demonstrate that Kinesin-Binding Protein (KBP) reduces the activity of KIF18A and KIF15 during metaphase. Overexpression of KBP disrupts the movement and alignment of mitotic chromosomes and decreases spindle length, a combination of phenotypes observed in cells deficient for KIF18A and KIF15, respectively. We show through gliding filament and microtubule co-pelleting assays that KBP directly inhibits KIF18A and KIF15 motor activity by preventing microtubule-binding. Consistent with these effects, the mitotic localizations of KIF18A and KIF15 are altered by overexpression of KBP. Cells depleted of KBP exhibit lagging chromosomes in anaphase, an effect that is recapitulated by KIF15 and KIF18A overexpression. Based on these data, we propose a model in which KBP acts as a protein buffer in mitosis, protecting cells from excessive KIF18A and KIF15 activity to promote accurate chromosome segregation.\n\nSUMMARYKinesin-Binding Protein (KBP) is identified as a regulator of the kinesins KIF18A and KIF15 during mitosis. KBP buffers the activity of these motors to control chromosome alignment and spindle integrity in metaphase and prevent lagging chromosomes in anaphase.

cell biology↗

bioRxiv2018

PIKfyve/Fab1 is required for efficient V-ATPase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum.\n\nPIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.\n\nImportanceCells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogen Legionella.

cell biology↗

bioRxiv2018

Mitotic chromosome alignment is required for proper nuclear envelope reassembly

Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell division, however, its importance to genomic stability and cellular fitness are not understood. Normal mammalian somatic cells lacking Kif18A function complete cell division without aligning chromosomes. These alignment-deficient cells display normal chromosome copy numbers in vitro and in vivo, suggesting that chromosome alignment is largely dispensable for maintenance of euploidy. However, we find that loss of chromosome alignment leads to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit, and the formation of micronuclei in vitro and in vivo. These defects slow cell proliferation and reduce postnatal growth and survival with variable penetrance in mice. Our studies support a model in which the alignment of mitotic chromosomes promotes proper nuclear envelope reassembly and continued proliferation by ensuring that chromosomes segregate as a compact mass during anaphase.

cell biology↗

bioRxiv2018

Tryptophan recovers sensitivity to cell membrane stress in Saccharomyces cerevisiae

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Because deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate, we implemented a suppressor gene screening revealing that the TAT2 tryptophan permease rescues cell growth to sodium dodecyl sulfate-treated{Delta} mck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that{Delta} trp1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as calcofluor white, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discover that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, our results expose a functional link between internal tryptophan levels and tryptophan biosynthesis in the response to plasma membrane damage.

cell biology↗

bioRxiv2018

Cardioprotection by the Mitochondrial Unfolded Protein Response is Mediated by ATF5

The mitochondrial unfolded protein response (UPRmt)1 is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. Activation of the UPRmt upregulates nuclear-encoded mitochondrial genes, including those for chaperones, proteases, and antioxidants, as well as glycolysis, to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5), a protein with both mitochondrial and nuclear targeting sequences, is proposed to mediate mammalian UPRmt signaling. Since proteostasis and bioenergetics are important in the response of organs such as the heart to injury, we hypothesized that pharmacologic UPRmt activation may be cardioprotective against ischemia-reperfusion (IR) injury and that such protection would require ATF5. Using a perfused heart IR injury model in wild-type and global Atf5-/- mice, we found that in-vivo administration of the UPRmt inducers oligomycin or doxycycline 6 h prior to ex-vivo IR injury was cardioprotective. Such protection was absent in hearts from Atf5-/- mice, and no protection was observed with acute ex-vivo cardiac administration of doxycycline. Loss of ATF5 also did not alter baseline IR injury (without UPRmt induction). Cardiac gene expression analysis by RNA-Seq revealed mild induction of numerous genes in an ATF5-dependent manner, which may be important for cardioprotection. Analysis of hearts by qPCR showed that oligomycin at 6 h significantly induced genes encoding ATF5 and several known UPRmt-linked proteins. We conclude that ATF5 is required for cardioprotection induced by drugs that activate the UPRmt.

cell biology↗

bioRxiv2018

The heterochromatin landscape in migrating cells and the importance of H3K27me3 for migration-associated transcriptional changes

H3K9me3, H3K27me3 and H4K20me1 are epigenetic markers associated with chromatin condensation and transcriptional repression. Previously, we found that migration of melanoma cells is associated with and dependent on global chromatin condensation that includes a global increase in these markers. Taken together with more recent reports by others suggest it is a general signature of migrating cells. Here, to learn about the function of these markers in migrating cells we mapped them by ChIP-seq analysis. This analysis revealed that induction of migration leads to expansion of these markers along the genome and to an increased overlapping between them. Significantly, induction of migration led to a higher increase in H3K9me3 and H4K20me1 signals at repetitive elements than at protein-coding genes, while an opposite pattern was found for H3K27me3. Transcriptome analysis revealed that 182 altered genes following induction of migration, of which 33% are dependent on H3K27me3 for these changes. H3K27me3 was also required to prevent changes in the expression of 501 other genes upon induction of migration. Taken together our results suggest that heterochromatinization in migrating cells is global and not restricted to specific genomic loci and that H3K27me3 is a key component in executing a migration-specific transcriptional plan.

cell biology↗

bioRxiv2018

Interrupted reprogramming into induced pluripotent stem cells does not rejuvenate human mesenchymal stromal cells

Replicative senescence hampers application of mesenchymal stromal cells (MSCs) because it limits culture expansion, impairs differentiation potential, and hinders reliable standardization of cell products. MSCs can be rejuvenated by reprogramming into induced pluripotent stem cells (iPSCs), which is associated with complete erasure of age- and senescence-associated DNA methylation (DNAm) patterns. However, this process is also associated with erasure of cell-type and tissue-specific epigenetic characteristics that are not recapitulated upon re-differentiation towards MSCs. In this study, we therefore followed the hypothesis that overexpression of pluripotency factors under culture conditions that do not allow full reprogramming might reset senescence-associated changes without entering a pluripotent state. MSCs were transfected with episomal plasmids and either successfully reprogrammed into iPSCs or cultured in different media with continuous passaging every week. Overexpression of pluripotency factors without reprogramming did neither prolong culture expansion nor ameliorate molecular and epigenetic hallmarks of senescence. Notably, transfection resulted in immortalization of one cell preparation with gain of large parts of the long arm of chromosome 1. Taken together, premature termination of reprogramming does not result in rejuvenation of MSCs and harbours the risk of transformation. This approach is therefore not suitable to rejuvenate cells for cellular therapy.

cell biology↗

bioRxiv2018

SAMICOS Determines Mitochondrial Cristae Architecture by Mediating mitochodrial Outer and Inner Membrane Contact

Mitochondrial cristae are critical for efficient oxidative phosphorylation, however, how cristae architecture is precisely organized remains largely unknown. Here, we discovered that Mic19, a core component of MICOS (mitochondrial contact site and cristae organizing system) complex, can be cleaved at N-terminal by mitochondrial protease OMA1. Mic19 directly interacts with mitochondrial outer-membrane protein Sam50 (the key subunit of SAM complex) and inner-membrane protein Mic60 (the key component of MICOS complex) to form Sam50-Mic19-Mic60 axis, which dominantly connects SAM and MICOS complexes to assemble MIB (mitochondrial intermembrane space bridging) supercomplex for mediating mitochondrial outer- and inner-membrane contact. OMA1-mediated Mic19 cleavage causes Sam50-Mic19-Mic60 axis disruption, which separates SAM and MICOS and leads to MIB disassembly. Disrupted Sam50-Mic19-Mic60 axis, even in the presence of SAM and MICOS complexes, causes the abnormal mitochondrial morphology, loss of mitochondrial cristae junctions, abnormal cristae distribution and reduced ATP production. Importantly, Sam50 displays punctate distribution at mitochondrial outer membrane, and acts as an anchoring point to guide the formation of mitochondrial cristae junctions. Therefore, we propose a model that Sam50-Mic19-Mic60 axis mediated SAM-MICOS complexes integration determines mitochondrial cristae architecture.

cell biology↗

bioRxiv2018

Effects of Human Sex Chromosome Dosage on Spatial Chromosome Organization

Sex chromosome aneuploidies (SCAs) are common genetic syndromes characterized by the presence of an aberrant number of X and Y chromosomes due to meiotic defects. These conditions impact structure and function of diverse tissues, but the proximal effects of SCA on genome organization are unknown. Here, to determine the consequences of SCAs on global genome organization, we have analyzed multiple architectural features of chromosome organization in a comprehensive set of primary cells from SCA patients with various ratios of X and Y chromosomes by use of imaging-based high-throughput Chromosome Territory Mapping (HiCTMap). We find that X chromosome supernumeracy does not affect the size, volume or nuclear position of the Y chromosome or an autosomal chromosome. In contrast, the active X chromosome undergoes architectural changes as a function of increasing X copy number, as measured by a decrease in size and an increase in circularity, which is indicative of chromatin compaction. With Y chromosome supernumeracy, Y chromosome size is reduced suggesting higher chromatin condensation. The radial positioning of chromosomes is unaffected in SCA karyotypes. Taken together, these observations document changes in genome architecture in response to alterations in sex chromosome numbers and point to trans-effects of dosage compensation on chromosome organization.

cell biology↗

bioRxiv2018

A combinatorial extracellular code tunes the intracellular signaling network activity to distinct cellular responses

Cells constantly survey a complex set of inputs that is processed by the intracellular signaling network, but little is known of how cells integrate input information from more than one cue. We employed a FRET biosensor-based imaging platform to study the effect of combinatorial growth factor levels on the signaling network in human cells. We found that pairwise stimuli caused distinct concentration- and ratio-dependent signaling states through signaling signatures such as antagonism, additivity and synergy. The unique signaling states correlated with differential gene expression and non-additive transcription patterns. We further elucidated how a signal-rich environment can fine-tune the signaling network and adjust physiological outcomes, by kinase and phosphatase activity profiling. We describe how complex extracellular conditions affect phospho-turnover and the basal phosphorylation status. Thus, we provide mechanistic insights into cellular processing of multiple cues and explain part of the complexity of cellular adaptation to changes in the extracellular environment.

cell biology↗

bioRxiv2018

Mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, are of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are only rescued by expressing wild-type nup88 and not the disease-linked mutant forms of nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

cell biology↗