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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,009 records · Page 56Linked to original sources

bioRxiv2018

Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity

It is known that the male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only 4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, here we investigate their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these cell-specific organelles are essential for the long-term post-mating behavior of females and that their formation is directly dependent upon Rab6. Our discovery adds to our understanding of Rab proteins function in secretory cells. We have created an online, open-access imaging resource as a valuable tool for the intracellular membrane and protein traffic community.

cell biology↗

bioRxiv2018

Octadecaneuropeptide prevents toxicity induced by 6-hydroxydopamine in cultured rat astrocyte: involvement of the endogenous antioxidant systems and the intrinsic apoptotic pathway

Oxidative stress, associated with various neurodegenerative diseases, induces imbalance in ROS generation, impairs cellular antioxidant defences and finally triggers both neurons and astroglial cell death by apoptosis. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN). We have previously reported that ODN is a potent neuroprotective agent that prevents 6-OHDA-induced apoptotic neuronal death. The purpose of the present study was to investigate the potential glioprotective effect of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Incubation of astrocytes with graded concentrations of ODN (10-14 to 10-8 M) inhibited 6-OHDA-evoked cell death in a concentration- and time-dependent manner. In addition, ODN prevented the decrease of mitochondrial activity and caspase-3 activation induced by 6-OHDA. Toxin-treated cells exhibited high level of ROS associated with a generation of H2O2 and O2{degrees}-and a reduction of both SOD and catalase activities. Co-treatment of astrocytes with low concentrations of ODN dose dependently blocked 6-OHDA-evoked ROS production and inhibition of antioxidant enzymes activities. Taken together, these data demonstrate that ODN is a potent glioprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death. ODN is thus a potential candidate to delay neuronal damages in various pathological conditions involving oxidative neurodegeneration.

cell biology↗

bioRxiv2018

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

cell biology↗

bioRxiv2018

Control of flagellum length by a grow-and-lock model

Several models have been proposed to explain how eukaryotic cells control the length of their cilia and flagella. Here, we investigated this process in the protist Trypanosoma brucei, an excellent system for cells with stable cilia like photoreceptors or spermatozoa. We show that the total amount of intraflagellar transport material (IFT, the machinery responsible for flagellum construction) increases during flagellum elongation, consistent with constant delivery of precursors and the previously reported linear growth. Reducing the IFT frequency by RNAi knockdown of the IFT kinesin motors slows down the elongation rate and results in the assembly of shorter flagella. These keep on elongating after cell division but fail to reach the normal length. This failure is neither due to an absence of precursors nor to a morphogenetic control by the cell body. We propose that the flagellum is locked after cell division, preventing further elongation or shortening. This is supported by the fact that subsequent increase in the IFT rate does not lead to further elongation. The distal tip FLAM8 protein was identified as a marker for the locking event. It is initiated prior cell division, leading to an arrest of elongation in the daughter cell. Here, we propose a new model termed grow-and-lock where the flagellum elongates until a locking event takes place in a timely defined manner hence fixing length. Alteration in the growth rate and/or in the timing of the locking event would lead to the formation of flagella of different lengths.

cell biology↗

bioRxiv2018

Role of the novel endoribonuclease SLFN14 and its disease causing mutations in ribosomal degradation

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

cell biology↗

bioRxiv2018

Cell atlas of human uterus

The human uterus is a highly dynamic tissue that undergoes repeated damage repair and regeneration during the menstrual cycle, which make it ideal model to study tissue regeneration and pathological process. Stem/progenitors were speculated to be involved in the regeneration of endometrial epithelial and pathogenesis of endometriosis. But the identity, microenvironment and regulatory mechanisms of the uterus epithelial stem/progenitors in vivo remain unclear. Here, we dissected the cell heterogeneities of the full-thickness human uterus epithelial cells (11 clusters), stroma cells (6 clusters), endothelial cells (5 clusters), smooth muscle cells (2 clusters), myofibroblasts (2 clusters) and immune cells (6 clusters) from 2735 single cell by single cell RNA-seq. Further analysis identified a unique ciliated epithelial cell cluster showing characteristics of stem/progenitors with properties of epithelial-mesenchymal transition (EMT) that mainly localized in the upper functionalis of the endometrium. Ordering the cell subpopulations along the pseudo-space revealed cell clusters possess cellular states of stress, inflammation and apoptosis in the upper functionalis cellular ecosystem of the endometrium. Connectivity map between the human uterus subpopulations revealed potential inflammatory (cytokines and chemokines) and developmental (WNT, FGF, VEGF) signals within the upper functionalis cellular ecosystem of the endometrium, especially from other epithelial clusters, regulating cell plasticity of the EMT-epithelial clusters. This study reconstructed the heterogeneities, space-specific distribution and connectivity map of human uterus atlas, which would provide insight in the regeneration of uterus endometria and reference for the pathogenesis of uterus.

cell biology↗

bioRxiv2018

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analyzed in detail and showed an intensive labeling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 labeling in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.\n\nSignificance statementCholesterol is a vital steroid molecule with many important functions in animal cells. Although its dysregulation is associated with an expanding list of clinically important pathologies, the study of its role is limited by a lack of reliable tools for live intracellular monitoring. This study demonstrates the applicability of a novel class of heterocyclic sterol probes. These probes exhibit fast cellular uptake with effective fluorescence labeling of sterol species in a variety of living cells, without a need for artificial carriers. When applied to Niemann-Pick disease type C1 cells, they identified massive accumulation of cholesterol in the endosome/lysosome compartment. Thus, several probes from the same series can also be used for visualizing lysosomal storage disorders and sterol transporting pathologies.

cell biology↗

bioRxiv2018

Direct measurement of myofilament calcium in living cardiomyocytes

Visualising when and where calcium appears and disappears in cardiomyocytes is a major goal of cardiovascular research. Surprisingly we find that the chemical dyes widely used for this purpose disrupt cell contractility, due at least in part due to direct inhibition of the acto-myosin ATPase required to generate force. In order to improve calcium detection methods, we have developed a genetically encoded indicator that sits within the myofilament to directly visualise the changes occurring at the sarcomere. This tool improves on established chemical dyes and untargeted genetically encoded indicators for analysing small molecule modulators of myofilament-based calcium signalling. Importantly this is achieved without any measurable change in contractile function.

cell biology↗

bioRxiv2018

TRPV4 is the temperature-sensitive ion channel of human sperm

Ion channels control sperm fertilizing ability by triggering hyperactivated motility, which is regulated by membrane potential, intracellular pH, and cytosolic calcium. Previous studies unraveled three essential ion channels that regulate these parameters: 1) the Ca2+ channel CatSper, 2) the K+ channel KSper, and 3) the H+ channel Hv1. However, the molecular identity of an additional sperm Na+ conductance that mediates initial membrane depolarization and, thus, triggers downstream signaling events is yet to be defined. Here, we functionally characterize DSper, the Depolarizing Channel of Sperm, as the temperature-activated channel TRPV4. It is functionally expressed at both mRNA and protein levels, while other temperature-sensitive TRPV channels are not functional in human sperm. DSper currents are activated by warm temperatures and mediate cation conductance, that shares a pharmacological profile reminiscent of TRPV4. Together, these results suggest that TRPV4 activation triggers initial membrane depolarization, facilitating both CatSper and Hv1 gating and, consequently, sperm hyperactivation.

cell biology↗

bioRxiv2018

Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization

Abnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3s zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on neuronal survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.

cell biology↗

bioRxiv2018

Diffusion rather than IFT provides most of the tubulin required for axonemal assembly

Tubulin enters the cilia by diffusion and motor-based intraflagellar transport (IFT). The respective contributions of each route in providing tubulin for axonemal assembly are unknown. To attenuate IFT-based transport, we expressed modified GFP-tubulins in strains possessing IFT81 and IFT74 with altered tubulin binding sites. E-hook deficient GFP-{beta}-tubulin normally incorporated into the axonemal microtubules; its transport frequency was reduced by ~90% in control cells and essentially abolished when expressed in a strain possessing IFT81 with an incapacitated tubulin-binding site. Despite the strong reduction in IFT, the share of E-hook deficient GFP-{beta}-tubulin in the axoneme was only moderately reduced indicating that most axonemal tubulin (~80%) enters cilia by diffusion. While not providing the bulk of axonemal tubulin, we propose that motor-based IFT is nevertheless critical for ciliogenesis because it ensures high concentrations of tubulin near the ciliary tip promoting axonemal elongation.

cell biology↗

bioRxiv2018

Phospho-regulation of Atoh1 is required for plasticity of secretory progenitors and tissue regeneration

The intestinal epithelium is maintained by a small number of self-renewing stem cells in homeostasis. In addition committed progenitors can contribute to the functional stem cell compartment at a low level during homeostasis and substantially during regeneration following tissue damage. However the mechanism of, and requirement for, progenitor plasticity in mediating pathological response has not been demonstrated. Here we show that multisite phosphorylation of the transcription factor Atoh1 is required both for the contribution of secretory progenitors to the intestinal stem cell pool and for a robust regenerative response following damage. In lineage tracing experiments Atoh1+ cells (Atoh1(WT)CreERT2 mice) show stem cell activity by giving rise to multilineage intestinal clones both in the steady state and after tissue damage. Notably in the colonic epithelium a single generation of Atoh1+ progenitors sustains 1 in 15 stem cells. In an activating Atoh1(9S/T-A)CreERT2 line, the loss of phosphorylation sites on the Atoh1 protein promotes secretory differentiation and inhibits the contribution of these cells to self-renewal. Finally, in a chemical colitis model the Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenic capacity that impacts overall regenerative response of the epithelium. Thus progenitor plasticity plays an integral part in maintaining robust self-renewal in the intestinal epithelium and the balance between stem and progenitor fate behaviour is directly co-ordinated by Atoh1 multi-site phosphorylation.

cell biology↗

bioRxiv2018

Aurora B-mediated exclusion of HP1a from late-segregating chromatin prevents formation of micronuclei

Late-segregating acentric chromosomes pose a serious risk to genomic integrity when they are excluded from dividing daughter nuclei and form damage-prone micronuclei. Insight into the cellular mechanisms that prevent the formation of micronuclei from acentrics come from studies demonstrating that acentrics reincorporate into daughter telophase nuclei by passing through Aurora B kinase-dependent channels in the nuclear envelope of Drosophila neuroblasts. Here, we uncover a mechanism of nuclear envelope channel formation in which localized concentrations of Aurora B preferentially phosphorylate H3(S10) on heterochromatic acentrics and their associated DNA tethers. This phosphorylation event prevents HP1a from associating with heterochromatin and results in localized inhibition of nuclear envelope reassembly on endonuclease- and X-irradiation-induced acentrics and the main daughter nuclei at the sites of acentric entry to promote the formation of channels. Finally, we find that HP1a also specifies initiation sites of nuclear envelope reassembly on undamaged chromatin. Taken together, these results demonstrate that Aurora B-mediated regulation of HP1a-chromatin interactions plays a key role maintaining genome integrity by locally preventing nuclear envelope assembly and facilitating incorporation of late-segregating acentrics into daughter nuclei.

cell biology↗

bioRxiv2018

Endothelia extrude apoptotic cells to maintain a constant barrier

The vascular system is lined with endothelial cells that, although only existing in a single monolayer, are key in the regulation of vascular barrier function. One of the major challenges these cells face is a routine exposure to environmental stressors that can induce apoptosis. Uncontrolled apoptosis in the endothelial monolayer threatens the ability of the cells to maintain their barrier function, resulting in vascular dysfunction. Therefore, we sought to identify ways in which endothelia maintain a cohesive monolayer during apoptotic events. We found that endothelial cells fated die will undergo a process of apoptotic cellular extrusion, similar to what has been described in the epithelium. We further show that endothelial extrusion uses a conserved S1P-S1PR2-RhoA signaling pathway in order to induce the formation of an actin ring that contracts closed, forcing the dying cell out of the monolayer while simultaneously filling in the gap left behind. Thus, endothelial extrusion successfully removes an apoptotic cell before it compromises the monolayer, preserving the barrier function.

cell biology↗

bioRxiv2018

Cellular Migration May Exhibit Intrinsic Left-Right Asymmetries: A meta-analysis

The intracellular model of embryonic left-right (LR) asymmetry proposes that body laterality originates from intrinsic chiral properties of individual cells, and several recent studies identified consistent chirality in the behavior of cells in vitro. Here, we explored one prediction of the intrinsic asymmetry model: that LR asymmetries would be present in a wide range of mammalian cells, manifesting in the form of LR-biased migration toward an attractant. We mined data from published papers on galvanotaxis and chemotaxis and quantitatively analyzed the migration trajectories of adult somatic cells, stem cells, and cancer cells to determine whether they display significant consistent LR biases in their movements toward migration targets. We found that several cell types exhibited LR biases during galvanotaxis and chemotaxis, and that treatments inhibiting cytoskeletal remodeling or targeting ion channel activity both abolished these LR biases. While we cannot conclusively rule out the existence of subtle biasing cues in the apparatus of some of the studies, the analysis of this dataset suggests specific assays and cell types for further investigation into the chiral aspects of intrinsic cell behavior.\n\nFundingThis work was funded by the American Heart Association Established Investigator grant 0740088N and NIH grants R01-GM077425 (to ML) and NRSA grant 1F32GM087107 (to LNV). M.L. is also supported by the G. Harold and Leila Y. Mathers Charitable Foundation.

cell biology↗

bioRxiv2018

Phosphatidylethanolamine produced in the inner mitochondrial membrane is essential for yeast cytochrome bc1 complex function

Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1p). Deletion of Psd1, which is lethal in mice, compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. To test this, we re-wired PE metabolism in yeast by re-directing Psd1p to the outer mitochondrial membrane or the endomembrane system. Our biochemical and functional analyses identified the IMS as the greatest barrier for PE import and demonstrated that PE synthesis in the IM is critical for cytochrome bc1 complex (III) function. Importantly, mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7p, impaired complex III activity similar to PSD1 deletion. Collectively, these data demonstrate that PE made in the IM by Psd1p is critical to support the intrinsic functionality of complex III and establish one likely mechanism.

cell biology↗

bioRxiv2018

Neuronal receptors display cytoskeleton-independent directed motion on the plasma membrane

Directed transport of transmembrane proteins is generally believed to occur via intracellular transport vesicles. However, using single particle tracking in rat hippocampal neurons with a pH-sensitive quantum dot probe which specifically reports surface movement of receptors, we have identified a subpopulation of neuronal EphB2 receptors that exhibit directed motion between synapses within the plasma membrane itself. This receptor movement occurs independently of the cytoskeleton but is dependent on cholesterol and is regulated by neuronal activity.

cell biology↗

bioRxiv2018

TRPM7 residue S1269 mediates cAMP dependence of Ca2+ influx

The nonspecific divalent cation channel TRPM7 (transient receptor potential-melastatin-like 7) is involved in many Ca2+ and Mg2+-dependent cellular processes, including survival, proliferation and migration. TRPM7 expression predicts metastasis and recurrence in breast cancer and several other cancers. In cultured cells, it can induce an invasive phenotype by promoting Ca2+-mediated epithelial-mesenchymal transition. We previously showed that in neuroblastoma cells that overexpress TRPM7 moderately, stimulation with Ca2+-mobilizing agonists leads to a characteristic sustained influx of Ca2+. Here we report that sustained influx through TRPM7 is abruptly abrogated by elevating intracellular levels of cAMP. Using pharmacological inhibitors and overexpression studies we show that this blockage is mediated by the cAMP effector Protein Kinase A (PKA). Mutational analysis demonstrates that the Serine residue S1269, which is present proximal to the coiled-coil domain within the protein c-terminus, is responsible for sensitivity to cAMP.

cell biology↗