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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 595 records · Page 33Linked to original sources

bioRxiv2017

Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Crosslinking of IgE-bound Fc{varepsilon}RI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the Fc{varepsilon}RI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-Fc{varepsilon}RI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

cell biology↗

bioRxiv2017

ESCRT-III acts in scissioning new peroxisomes from the ER

Dynamic control of peroxisome proliferation is integral to the peroxisomes many functions. A breakdown in the ability of cells to form peroxisomes is linked to many human health issues, including defense against infectious agents, cancer, aging, heart disease, obesity and diabetes, and forms the basis of a spectrum of peroxisomal genetic disorders that cause severe neuropathologies. The ER serves as a source for preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and to support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. Here we show that the evolutionarily ancient endosomal sorting complexes required for transport (ESCRT)-III are peroxisome biogenesis factors that function to cleave PPVs budding from the ER into the cytosol. Using comprehensive morphological and genetic assays of peroxisome formation and function we find that absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay we show that ESCRT-III proteins Vps20 and Snf7 are required to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and towards the cytosol, a finding that has important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

cell biology↗

bioRxiv2017

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

Centrioles are composed of long-lived microtubules arranged in nine triplets. In unicellular eukaryotes, loss of the noncanonical tubulins, delta-tubulin and epsilon tubulin, result in loss of the triplet microtubule structure. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Here, we report the first characterization of delta-tubulin and epsilon-tubulin null human cells. Centrioles in cells lacking either delta-tubulin or epsilon-tubulin lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

cell biology↗

bioRxiv2017

Principles that govern competition or co-existence in Rho-GTPase driven polarization

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for uni-polar or multi-polar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We derive a \"saturation rule\" general to all relevant models that governs the timescale of competition, and thereby predicts whether the system will generate uni-polar or multi-polar outcomes. We suggest that the saturation rule is a fundamental property of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism.

cell biology↗

bioRxiv2017

aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts

Cell fate generation can rely on the unequal distribution of molecules during progenitor cell division in the nervous system of vertebrates and invertebrates. Here we address asymmetric fate determinant localization in the developing Drosophila nervous system, focussing on the control of asymmetric Miranda distribution in larval neuroblasts. We used live imaging of neuroblast polarity reporters at endogenous levels of expression to address Miranda localization during the cell cycle. We reveal that the regulation and dynamics of cortical association of Miranda in interphase and mitosis are different. In interphase Miranda binds directly to the plasma membrane. At the onset of mitosis, Miranda is phosphorylated by aPKC and displaced from the PM. After nuclear envelope breakdown asymmetric localization of Miranda requires actomyosin activity. Therefore, Miranda phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization.

cell biology↗

bioRxiv2017

NuMA Targets Dynein to Microtubule Minus-Ends at Mitosis

To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin, and both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends requires a C-terminal region outside NuMAs canonical microtubule binding domain, and it is independent of direct minus-end binders {gamma}-TuRC, CAMSAP1, and KANSL1/3. Both NuMAs minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

cell biology↗

bioRxiv2017

GADD45α regulates cell proliferation and DNA repair of BRL-3A cells that treated by FZD/UVC via P38, JNK, CDC2/CCNB1, AKT and MTOR pathways

GADD45 is a stress-induced gene activated by a variety of stress stimuli, including ultraviolet and ionizing radiation, and involved in cell cycle regulation, apoptosis, maintenance, genomic stability, DNA repair and immune response. However, the effects and regulatory mechanism of GADD45 on proliferation, apoptosis and DNA damage repair of hepatocytes in liver regeneration remains unclear. In this study overexpression of GADD45 significantly inhibited the cell viability, proliferation, the number of cells in G1 and S phases, and of furazolidone (FZD) or UVC induced apoptosis of BRL-3A cells and decreased the inhibition of FZD/UVC on the viability, proliferation of BRL-3A cells, while increased the number of cells in G2/M phase of BRL-3A cells and FZD/UVC induced S phase arrest. Downregulated GADD45 induced the viability, proliferation, the number of cells in S and G2/M phases and the inhibition of FZD/UVC on the viability, proliferation of BRL-3A cells increased, while decreased apoptosis, the number of cells in G1 phases of BRL-3A cells and FZD/UVC induced S phase arrest. The results of qRT-PCR and western blot showed that genes/proteins related to P38MAPK, JNK, CDC2/CCNB1, AKT and MTOR signaling pathways were significantly changed in normal BRL-3A cells. The expression profiles of cell cycle, proliferation and apoptosis related genes/proteins in FZD/UVC treated BRL-3A cells were also detected by qRT-PCR and western blot, and the results indicated that the expression of Myc, Bcl-2, Ccnd1, PCNA, P21, Ccnb1, Caspase3, Caspase8, Caspase9 and Bax have significantly changes.

cell biology↗

bioRxiv2017

Exosomes from Nef expressing monocytic cells restrict HIV-1 replication in infected cells through the assembly of stress granules

Exosomes are membranous vesicles secreted from almost all types of cells, carry proteins and nucleic acids and function as vehicles for intercellular communication. Cells infected with HIV-1 or expressing the viral Nef protein secrete more exosomes than uninfected cells or those not expressing this protein. We used stably transfected, Nef-expressing U937 human monocytic cells and exosomes purified from these cells to study their effects on HIV-1 infected and uninfected CD4+ T-cells. The Nef exosomes inhibited virus production from HIV-1 infected CD4+ T-cells, but caused activation induced cell death in uninfected bystander cells. Mutations in its conserved Arginine residues and in the secretion-modification-region failed to secrete Nef into exosomes. Cell lines expressing these mutant Nef proteins did not deliver it to the target CD4+ T-cells, and exosomes prepared from these mutant Nef-expressing cells also did not inhibit virus production. Nef exosomes inhibited virus production by inducing the assembly of stress granules in HIV-1 infected cells, which sequestered increased amounts of gag mRNA. This is a novel mechanism wherein we show the effects of exosomes on the assembly of stress granules and viral translational repression.

cell biology↗

bioRxiv2017

Akt/PKB enhances non-canonical Wnt signals by compartmentalizing β-Catenin

Cellular proliferation is antagonistically regulated by canonical and non-canonical Wnt signals; their dysbalance triggers cancers. It is widely believed that the PI3-K[->] Akt pathway enhances canonical Wnt signals by affecting transcriptional activity and stability of {beta}-catenin. Here we demonstrate that the PI3-K[->]Akt pathway also enhances non-canonical Wnt signals by compartmentalizing {beta}-catenin. By phosphorylating the phosphoinositide(PI)-binding domain of a multimodular signal transducer, Daple, Akt abolishes Daples ability to bind PI3-P-enriched endosomes that engage dynein motor complex for long-distance trafficking of {beta}-catenin/E-cadherin complexes to pericentriolar recycling endosomes (PCREs). Phosphorylation compartmentalizes Daple/{beta}-catenin/E-cadherin complexes to cell-cell contact sites, enhances non-canonical Wnt signals, and thereby, suppresses colony growth. Dephosphorylation compartmentalizes {beta}-catenin on PCREs, a specialized compartment for prolonged unopposed canonical Wnt signaling, and enhances colony growth. Cancer-associated Daple mutants that are insensitive to Akt mimic a constitutively dephosphorylated state. This work not only identifies Daple as a platform for crosstalk between Akt and the non-canonical Wnt pathway, but also reveals the impact of such crosstalk during cancer initiation and progression.

cell biology↗

bioRxiv2017

EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

It is not clear why central nervous system (CNS) axons lose their intrinsic ability to regenerate with maturity, whilst peripheral (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their rab11 positive carriers. We reasoned that this exclusion might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this hypothesis using laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 GEF EFA6. EFA6 exerts its effects from a previously unreported location within the axon initial segment (AIS). EFA6 does not localise here in DRG axons, and in these neurons, ARF activation is counteracted by an ARF-GAP which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whilst overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the axon initial segment, signalling and transport.\n\nSummary StatementWe identify a novel resident of the axon initial segment, EFA6. This functions to prevent growth-promoting molecules from entering mature CNS axons. Removing EFA6 elevates the axons regenerative potential.

cell biology↗

bioRxiv2017

Multi-BRCT domain protein Brc1 links Rhp18/Rad18 and γH2A to maintain genome stability during S-phase

DNA replication involves the inherent risk of genome instability, as replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during S-phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phospho-histone H2A ({gamma}H2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1-4 of Brc1 have remained obscure. Here we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and {gamma}H2A are required for the Brc1-overexpression suppression of smc6-74, which impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings we propose that Brc1 provides scaffolding functions linking {gamma}H2A, Rhp18, and Smc5/6 complex at damaged replication forks.

cell biology↗

bioRxiv2017

The SMuSh pathway is essential for survival during growth-induced compressive mechanical stress

Cells that proliferate within a confined environment build up mechanical compressive stress. For example, mechanical pressure emerges in the naturally space-limited tumor environment. However, little is known about how cells sense and respond to mechanical compression. We developed microfluidic bioreactors to enable the investigation of the effects of compressive stress on the growth of the genetically tractable model organism Saccharomyces cerevisiae. We used this system to determine that compressive stress is partly partly sensed through a module consisting of the mucin Msb2, and the cell wall protein Sho1, which act together as a sensor module in one of the two major osmosensing pathways in budding yeast. This signal is transmitted via the MAPKKK kinase Ste11. Thus, we term this mechanosensitive pathway the SMuSh pathway, for Ste11 through Mucin / Sho1 pathway. The SMuSh pathway delays cells in the G1 phase of the cell cycle and improves cell survival in response to growth-induced pressure. We also found that the Cell Wall Integrity (CWI) pathway contributes to the response to mechanical compressive stress. These latter results are confirmed in complimentary experiments in the accompanying manuscript from Mishra et al. When both the SMuSh and the CWI pathways are deleted, cells fail to adapt to compressive stress and all cells lyse at relatively low pressure when grown in confinement. Thus, we define a network that is essential for cell survival during growth under pressure. We term this new mechanosensory system the SCWISh (Survival through the CWI and SMuSh) network.\n\nSignificance StatementGrowth in confined environments leads to the build up of compressive mechanical stresses, which are relevant to diverse fields, from cancer to microbiology. In contrast to tensile stress, little is known about the molecular integration of compressive stresses. In this study, we elucidate the SMuSh pathway, which, together with the Cell Wall Integrity pathway, is essential for viability of the budding yeast S. cerevisiae when growing under mechanical pressure. Pressure-sensing requires the transmembrane mucin, Msb2, which is linked to the actin cortex. Our result raises the intriguing question of whether mucins, widely conserved in eukaryotes and frequently misregulated in cancers, might sense compressive stresses in other organisms, including humans.

cell biology↗

bioRxiv2017

Crosslinkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

Cytokinesis and other cell shape changes are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in non-muscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal crosslinking by myosins and non-motor crosslinkers, are thought to promote contractility. Here, we examined how the abundance of motor and non-motor crosslinkers controls the speed of cytokinetic furrowing. We built a minimal model to simulate the contractile dynamics of the C. elegans zygote cytokinetic ring. This model predicted that intermediate levels of non-motor crosslinkers would allow maximal contraction speed, which we found to be the case for the scaffold protein anillin, in vivo. Our model also demonstrated a non-linear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of non-muscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, both motor and non-motor crosslinkers promote cytokinetic ring closure when present at low levels, but act as a brake when present at higher levels. Together, our findings extend the growing appreciation for the roles of crosslinkers, but reveal that they not only drive but also brake cytoskeletal remodeling.

cell biology↗

bioRxiv2017

Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex, the core component of the kinetochore-microtubule interface, to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

cell biology↗

bioRxiv2017

Distinct roles of the polarity factors Boi1 and Boi2 in the control of exocytosis and abscission in budding yeast

Boi1 and Boi2 (Boi1/2) are budding yeast plasma membrane proteins that function in polarized growth, and in cytokinesis inhibition in response to chromosome bridges via the NoCut abscission checkpoint. How Boi1/2 act in these two distinct processes is not understood. We demonstrate that Boi1/2 are required for a late step in the fusion of secretory vesicles with the plasma membrane of the growing bud. Cells lacking Boi1/2 accumulate secretory vesicles and are defective in bud growth. In contrast, Boi2 is specifically required for abscission inhibition in cells with chromatin bridges. The SH3 domain of Boi2, which is dispensable for bud growth and targets Boi2 to the site of abscission, is essential for abscission inhibition. Gain of function of the exocyst, a conserved protein complex involved in tethering of exocytic vesicles to the plasma membrane, rescued secretion and bud growth defects in boi mutant cells, and abrogated NoCut checkpoint function. Thus, Boi2 functions redundantly with Boi1 to promote the fusion of secretory vesicles with the plasma membrane at sites of polarized growth, and acts as an abscission inhibitor during cytokinesis in response to chromatin bridges.

cell biology↗

bioRxiv2017

Cargo crowding at actin-rich regions along axons causes local traffic jams in neurons

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary across a broad distribution of times. We examine the transport of pre-synaptic vesicles (pre-SVs), endosomes and mitochondria in C. elegans touch receptor neurons (TRNs), showing that stalled cargo are predominantly present at actin-rich regions along the neuronal process. Cargo stalled at actin-rich regions increase the propensity of moving cargo to stall at the same location, resulting in traffic jams. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they occur in Drosophila neurons as well. These traffic jams can act as both sources and sinks of vesicles. We propose that they act as functional reservoirs that contribute to maintaining robust cargo flow in the neuron.

cell biology↗

bioRxiv2017

Cross-inhibition of JNK by p38 visualized by multiplexed fluorescence imaging

AbstractThe stress activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and p38, are important players in cell fate decisions in response to environmental stress signals. Crosstalk signaling between JNK and p38 is emerging as an important regulatory mechanism in the inflammatory and stress responses. However, it is still unknown how this crosstalk affects signaling dynamics, cell-to-cell variation, and cellular responses at the single-cell level. To address these questions, we established a multiplexed live-cell imaging system based on kinase translocation reporters to simultaneously monitor JNK and p38 activities with high specificity and sensitivity at single-cell resolution. Various stresses, such as anisomycin, osmotic stress, and UV irradiation, and pro-inflammatory cytokines activated JNK and p38 with various dynamics. In all cases, however, p38 suppressed JNK activity in a cross-inhibitory manner. We demonstrate that p38 antagonizes JNK through both transcriptional and post-translational mechanisms. This cross-inhibition of JNK appears to generate cellular heterogeneity in JNK activity after stress exposure. Our data indicate that this heterogeneity in JNK activity plays a role in fractional killing in response to UV stress. Our highly sensitive multiplexed imaging system enables detailed investigation into the p38-JNK interplay in single cells.\n\nOne Sentence SummaryCross-inhibition by p38 generates cell-to-cell variability in JNK activity.

cell biology↗

bioRxiv2017

A urine-dependent human urothelial organoid offers a viable alternative to rodent models of infection

Murine models describe a defined host/pathogen interaction for urinary tract infection, but human cell studies are scant. Although recent human urothelial organoid models are promising, none demonstrate long-term tolerance to urine, the natural substrate of the tissue and of the uropathogens that live there. We developed a novel human organoid from progenitor cells which demonstrates key structural hallmarks and biomarkers of the urothelium. After three weeks of transwell culture with 100% urine at the apical interface, the organoid stratified into multiple layers. The apical surface differentiated into enlarged and flattened umbrella-like cells bearing characteristic tight junctions, structures resembling asymmetric unit membrane plaques, and a glycosaminoglycan layer. The apical cells also expressed apical cytokeratin-20, a spatial feature of the mammalian urothelium. Urine itself was necessary for full development, and undifferentiated cells were urine-tolerant despite the lack of membrane plaques and a glycosaminoglycan layer. Infection with Enterococcus faecalis revealed the expected invasive outcome, including urothelial sloughing and the formation of intracellular colonies similar to those previously observed in patient cells. This new biomimetic model could help illuminate invasive behaviours of uropathogens, and serve as a reproducible test bed for disease formation, treatment and resolution in patients.

cell biology↗