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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

An ancestral role of pericentrin in centriole formation through SAS-6 recruitment

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.\n\nShort summaryThe pericentriolar matrix (PCM) is not only important for microtubule-nucleation but also can regulate centriole biogenesis. Ito et al. reveal an ancestral interaction between the centriole protein SAS-6 and the PCM component pericentrin, which regulates centriole biogenesis and elongation.

cell biology↗

bioRxiv2018

Nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through regulating AKT/mTOR/S6K/4EBP1 signaling pathway

Migration and contraction of activated hepatic stellate cell (HSC) are essential factors for cirrhosis formation and development. It has been demonstrated that blebbistatin, a nonmuscle myosin II (NMMII) inhibitor, can inhibit the migration and contraction of HSC, whereas the main cell signaling pathway is still unknown. Mammalian target of rapamycin (mTOR) signaling pathway may be involved in many cells migration and contraction, whether NMMII and mTOR have any crosslinks draw our attention. In the currently study, we used LV-RNAi to specifically attenuate mTOR and NMMII in rat HSC. We aimed to examine the effect of mTOR LV-RNAi on the migration and contraction of HSC and explore the crosslink between mTOR cell signal and NMMII. Using real-time PCR and western blot, we found that mTOR and the downstream factors including S6K and 4EBP1 all up-regulated with the activation of HSC, mTOR and NMMII LV-RNAi was transfected into activated HSC using lipofectamine 2000. The levels of mRNA and proteins were also examined using real-time PCR and western blot respectively. The expression of mTOR can be down-regulated by NMMII LV-RNAi significantly, as well as the expression of S6K, 4EBP1, -SMA and collagen I, but the level of AKT was up-regulated. Then we used Transwell system and collagen lattices to examine the NMMII and mTOR LV-RNAi efficiency on HSC migration and contraction, as we hypothesized, both of the LV-RNAi could inhibit HSC migration and contraction significantly. These results indicated that nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through the regulation of mTOR/S6K/4EBP1 signaling pathway

cell biology↗

bioRxiv2018

Self-organization of Plk4 regulates symmetry breaking in centriole duplication.

During centriole duplication, a single daughter centriole is formed near the mother centriole. The mechanism that determines a single duplication site is unknown. Here, we demonstrate that intrinsic self-organization of Plk4 underlies symmetry breaking in centriole duplication. We show that in its nonphosphorylated state, Plk4 preferentially self-assembles via a disordered linker and that this self-assembly is prevented by autophosphorylation. Consistently, the dissociation dynamics of centriolar Plk4 are controlled by autophosphorylation. We further found that autophophorylated Plk4 is localized as a single focus around the mother centriole before procentriole formation, and is subsequently targeted for STIL-HsSAS6 loading. Perturbing Plk4 self-organization affects the asymmetry of centriolar Plk4 distribution and centriole duplication. We propose that the spatial patterning of Plk4 directs a single duplication site per mother centriole.

cell biology↗

bioRxiv2018

ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT) machinery are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX. Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens.

cell biology↗

bioRxiv2018

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

cell biology↗

bioRxiv2018

Plate-based assay of AP-3 traffic in budding yeast

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening. SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathways importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

cell biology↗

bioRxiv2018

LRH-1 Mitigates Intestinal Inflammatory Disease by Maintaining Epithelial Homeostasis and Cell Survival

Epithelial dysfunction and loss of intestinal crypts are defining features of inflammatory bowel disease (IBD). However, current therapies primarily target the immune system and not the epithelium. The nuclear receptor LRH-1 encoded by Nr5a2 is expressed in intestinal epithelium and is thought to contribute to epithelial renewal. Here we investigate how loss and gain of LRH-1 impacts the intestinal epithelium in healthy and inflammatory conditions. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death and weakens the epithelial barrier. Loss of LRH-1 also distorts the cellular composition of the epithelium, resulting in an expansion of Paneth and goblet cells, and a decrease in enteroendocrine cells. Human LRH-1 (hLRH-1) not only rescues epithelial integrity, but when overexpressed, mitigates inflammatory damage in mouse and human intestinal organoids, including those from IBD patients. Finally, hLRH-1 greatly reduces disease severity in a mouse model of T cell-mediated colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNF-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.

cell biology↗

bioRxiv2018

Comparison of antioxidant activity between cyanidin-3-O-glucoside (C3G) liposome and cyanidin-3-O-glucoside (C3G) in 2D and 3D cell cultures

The 2D cell culture is the predominant in vitro model for numerous studies. However, 2D cell cultures may not accurately reflect the functions of three-dimensional (3D) tissues, which have extensive cell-cell and cell-matrix interactions; thus, using 2D cell cultures may lead to inaccurate experimental results. Therefore, to obtain adequate and detailed information about the antioxidant activity of cyanidin-3-O-glucoside (C3G) and C3G liposomes in the 2D and 3D cell culture models, we used in this study H2O2 to construct the cell damage model and assess the antioxidant activity of C3G and C3G liposomes on Caco-2 cells cultured in the 3D model. We also measured the cell viability, cell morphology, and activity of glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content of Caco-2 cells treated with H2O2, C3G, and C3G liposomes. Results showed that cells cultured in the 3D culture model formed a 3D structure and tight spheroids and showed increased cell activity and IC50. The C3G and C3G liposomes can enhance the activity of GSH, SOD, and T-AOC but decrease the MDA content. At the same time, the effect was more obvious in the 3D cell culture model than in the cells cultured in the 2D model. This study revealed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. A realistic mechanism study of antioxidant activity of C3G and C3G liposomes in the 3D cell model, which acts as an intermediate stage bridging the in vitro 2D and in vivo models, was observed.

cell biology↗

bioRxiv2018

Enterovirus 71 structural viral protein 1 promotes mouse Schwann cell autophagy via endoplasmic reticulum stress-mediated peripheral myelin protein 22 upregulation

Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease-related deaths due to fatal neurological complications. The clinical observations and animal models found the early invasion of nervous system, and the demyelinating phenomenon was observed. As one of the receptors of EV71 structural viral protein 1 (VP1), SCARB2 mainly exists on the myelin sheath. EV71 VP1 can promote viral replication through inducing autophagy in neuron cells. This study aims to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells (MSCs). An EV71 VP1-expressing vector (pEGFP-C3-VP1) was generated and transfected into MSCs. Transmission electron microscopy (TEM) and Western blot analysis of the autophagy marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) were used to assess autophagy in the cells. Real-time PCR and immunofluorescent staining were performed to determine the expression of PMP22. Small interfering RNA against PMP22 was employed to investigate the role of PMP22 in MSCs autophagy. Selective endoplasmic reticulum (ER) stress inhibitor salubrinal (SAL) was employed to determine whether PMP22 is mediated by ER stress. Our results demonstrated that VP1 played a promotive role in MSC autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3B and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by SAL. VP1 promotes MSC autophagy through upregulating ER stress-mediated PMP22 expression. VP1/ER stress/ PMP22 axis in autophagy may be a potential therapeutic target for EV71 infection-induced fatal neuronal damage.

cell biology↗

bioRxiv2018

Direct reprogramming of human epithelial cells into organoids by miR-106a-3p

The 3D cultures provide more insight into cell-to-cell and cell-to-matrix interactions, better mimicking the environment where stem cells reside compared to traditional 2D cultures. Although the precise molecular pathways involved in the regulation of stem and progenitor cell fate remain unknown, it is widely accepted that transcription factors play a crucial role as intrinsic regulators in these fate decisions. In this study, we carried out a microRNA screen to track the behaviour of adult stem/progenitor cells derived from human mammary epithelial cells grown in 3D cultures. We identified miR-106a-3p, which enriches the adult stem cell-like lineage and promotes the expansion of 3D cultures. Transcriptomic analysis showed that this miRNA regulates transcription factors such as REST, CBFB, NF-YA, and GATA3, thereby enhancing the maintenance of adult stem/progenitor cells in human epithelial cells. These data reveal a clear transcriptional program that governs the maintenance of adult stem/progenitor cells and controls their fate.

cell biology↗

bioRxiv2018

Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in Drosophila melanogaster

Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. Here, we report a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. We show that reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. We also report that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. Our results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation.\n\nBrief summary statementThe authors show that the membrane phospholipid PIP2, and the kinase that produces PIP2 called Skittles, are needed for normal ciliary transition zone morphology and function in the Drosophila male germline.

cell biology↗

bioRxiv2018

Inward tubulation of the plasma membrane expedites membrane exchange and receptor presentation

The plasma membrane is a crucial barrier between the cell and its external environment, and it also enables efficient passage of materials and information. Membrane vesicle trafficking allows precise delivery of materials but is rather inefficient. The mechanism for efficient membrane exchange remains elusive. Here we describe inward tubulation of the plasma membrane (PM tubes) that extends deep into the cytoplasm. These widespread PM tubes elongate along microtubules and are stabilized by actin filaments and cholesterol. PM tubes are preferred sites for connection between the endoplasmic reticulum and the plasma membrane. PM tubes facilitate receptor presentation at the surface of cells, possibly also shortening the distance for transported cargo to reach the external environment.\n\nIn BriefA new type of tubular membrane structures was discovered in cells, revealing a shortcut that cells employ to expedite material exchange with their external environment.\n\nHighlightsO_LIInward tubulation of the plasma membrane (PM tubes), transiently interacts with the Golgi apparatus\nC_LIO_LIMicrotubule side-binding proteins pull PM tubes, while actin filaments and cholesterol stabilize PM tubes\nC_LIO_LIPM tubes are preferred sites where ER-PM contacts form in response to increased cytoplasmic calcium concentration\nC_LIO_LIPM tubes are preferred sites for the surface presentation of GLUT1 upon glucose deprivation\nC_LI

cell biology↗

bioRxiv2018

MEF2C is a new regulator of the human articular chondrocyte phenotype

MEF2C plays a role in diverse tissues, most notably heart, brain, eyes and developing bones. Here we report for the first time that MEF2C is present and active in the permanent articular cartilage in humans which lines and protects our joints throughout life. We show that MEF2C directly targets cartilage master regulator gene SOX9, and SOX9, in turn, regulates MEF2C in a novel positive feedback loop maintaining high expression levels of both transcription factors, and consequently stabilising the articular chondrocyte phenotype and helping prevent hypertrophy and subsequent calcification and vascularisation. We propose that MEF2C and SOX9 may show similar cooperative activity in other tissues, and across a range of adult murine tissues we found co-expression of both transcription factors in cartilage, trachea, brain, eyes and heart. Strikingly, all of these tissues are prone to calcification and further study of MEF2C/SOX9 cooperativity in these organs will be revealing.

cell biology↗

bioRxiv2018

A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which give rise to micronucleated daughter cells.

cell biology↗

bioRxiv2018

Chimeric antigen receptors that trigger phagocytosis

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity, and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcR{gamma} robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing macrophages reduce cancer cell number in co-culture by over 40%.\n\nSummaryWe report the first Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that promote engulfment of antigen-coated particles and cancer cells.

cell biology↗

bioRxiv2018

Fission yeast cytokinesis requires a putative mechanosensitive channel Pkd2p

Force plays a central role in separating daughter cells during cytokinesis, the last stage of cell division. However, the mechanism of force-sensing during cytokinesis remains unknown. Here we discovered that Pkd2p, a putative force-sensing TRP channel, localizes to the cleavage furrow during cytokinesis of the fission yeast, Schizosaccharomyces pombe. Pkd2p, whose human homologues are associated with Autosomal Polycystic Kidney Disease, is an essential protein whose localization depends on the contractile ring and the secretory pathway. We identified and characterized a novel pkd2 mutant pkd2-81KD. The pkd2 mutant cells show signs of osmotic stress, including temporary shrinking, paused turnover of the cytoskeletal structures and hyper-activated MAPK signaling. During cytokinesis, although the contractile ring constricts more rapidly in the pkd2 mutant than the wild-type cells (50% higher), the cell separation in the mutant is slower and often incomplete. These cytokinesis defects are also consistent with mis-regulated turgor pressure. Lastly, the pkd2 mutant exhibits strong genetic interactions with two mutants of the SIN pathway, a signaling cascade essential for cytokinesis. We propose that Pkd2p modulates osmotic homeostasis and is potentially a novel regulator of cytokinesis.\n\nHighlight summary for TOCFission yeast TRP channel Pkd2p is the homologue of human polycystins. The pkd2 mutant exhibits defects in the contractile ring closure and cell separation during cytokinesis. This essential protein localizes to the cleavage furrow where it likely regulates osmotic homeostasis during cytokinesis.

cell biology↗

bioRxiv2018

A Composition-Dependent Molecular Clutch Between T Cell Signaling Clusters and Actin

Biomolecular condensates play important roles in eukaryotic cells by concentrating molecules into foci without a surrounding membrane. During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of the adaptor protein LAT and its binding partners Grb2, Sos1, SLP-76, Nck and WASP. These condensates move radially at the IS, traversing a radially-oriented and then a concentric actin network. To understand the persistent radial movement, we biochemically reconstituted LAT condensates with mobile actomyosin filaments. We found that basic regions of Nck and N-WASP promote strong association and co-movement of LAT condensates with actin. Condensates lacking these components were instead propelled by steric interactions. In cells, LAT condensates lost Nck while traversing the boundary between the two actin networks, and condensates engineered to constitutively bind actin moved aberrantly. We propose that Nck and WASP form a clutch between LAT condensates and actin, and changes in composition enable condensate movement by distinct actin networks in different regions of the IS.

cell biology↗

bioRxiv2018

Automated Tracking of Biopolymer Growth and Network Deformation with TSOAX

Studies of how individual semi-flexible biopolymers and their network assemblies change over time reveal dynamical and mechanical properties important to the understanding of their function in tissues and living cells. Automatic tracking of biopolymer networks from fluorescence microscopy time-lapse sequences facilitates such quantitative studies. We present an open source software tool that combines a global and local correspondence algorithm to track biopolymer networks in 2D and 3D, using stretching open active contours. We demonstrate its application in fully automated tracking of elongating and intersecting actin filaments, detection of loop formation and constriction of tilted contractile rings in live cells, and tracking of network deformation under shear deformation.

cell biology↗