SEARCH · Search bioRxiv

Results for “Cell Biology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources

bioRxiv2016

Temporal regulation of epithelium formation

Abstract/SummaryTo establish the animal body plan, embryos link the external epidermis to the internal digestive tract. In Caenorhabditis elegans, this linkage is achieved by the Arcade Cells, which form an epithelial bridge between the foregut and epidermis, but little is known about how development of these three epithelia is coordinated temporally. The Arcade Cell epithelium is generated after the epidermis and digestive tract epithelia have matured, ensuring that both organs can withstand the mechanical stress of embryo elongation; mis-timing of epithelium formation leads to defects in morphogenesis. Here, we report that temporal regulation of the Arcade Cell epithelium is mediated by the pioneer transcription factor PHA-4/FoxA, the cytoskeletal regulator ZEN-4/MKLP and the polarity protein PAR-6. We find that PHA-4 activates expression of a broad cohort of epithelial genes. However, accumulation of protein is delayed by ZEN-4, acting in concert with its partner CYK-4/MgcRacGAP. Finally, PAR-6 localizes factors within adherens junctions and at the apical surface, leading to Arcade Cell polarity. The results reveal that the timing of a landmark event during embryonic morphogenesis is mediated by the concerted action of four proteins that delay the formation of an epithelial bridge. In addition, we find that FoxA associates with many epithelial genes in mammals, suggesting that regulation of epithelial identity may be a conserved feature of FoxA factors and a contributor to FoxA function in development and cancer.

Cell Biology↗

bioRxiv2016

ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics

Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, {gamma}-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through {gamma}-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

Cell Biology↗

bioRxiv2016

Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

Niemann Pick C disease (NPC) is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation NPC1I1061T has been shown to cause endoplasmic reticulum associated degradation of the NPC1 protein. Treatment of patient derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACi) Vorinostat or Panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Herein we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by Vorinostat or Panobinostat and that treatment with Vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 81 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 80% of the mutants showed reduced cholesterol accumulation when treated with Vorinostat or Panobinostat.

Cell Biology↗

bioRxiv2016

Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

The spatial organization of eukaryotic genomes is non-random, cell-type specific, and has been linked to cellular function. The investigation of spatial organization has traditionally relied extensively on fluorescence microscopy. The validity of the imaging methods used to probe spatial genome organization often depends on the accuracy and precision of distance measurements. Imaging-based measurements may either use 2 dimensional datasets or 3D datasets including the z-axis information in image stacks. Here we compare the suitability of 2D versus 3D distance measurements in the analysis of various features of spatial genome organization. We find in general good agreement between 2D and 3D analysis with higher convergence of measurements as the interrogated distance increases, especially in flat cells. Overall, 3D distance measurements are more accurate than 2D distances, but are also more prone to noise. In particular, z-stacks are prone to error due to imaging properties such as limited resolution along the z-axis and optical aberrations, and we also find significant deviations from unimodal distance distributions caused by low sampling frequency in z. These deviations can be ameliorated by sampling at much higher frequency in the z-direction. We conclude that 2D distances are preferred for comparative analyses between cells, but 3D distances are preferred when comparing to theoretical models in large samples of cells. In general, 2D distance measurements remain preferable for many applications of analysis of spatial genome organization.

Cell Biology↗

bioRxiv2016

The appressorium of Magnaporthe oryzae remains mitotically active during post-penetration hyphal growth in rice cells

To investigate the mitotic dynamics of an appressorium, we used time-lapse confocal imaging of a fluorescence-based mitotic reporter strain of Magnaporthe oryzae. We present evidence that: (i) appressoria remain viable and mitotically active after host penetration, (ii) appressorial mitosis, like invasive hyphal mitosis, is semi-closed, (iii) sister chromatids separate within the appressorium, (iv) a mitotic appressorial nucleus undergoes extreme constriction and elongation as it migrates through the penetration peg in a manner analogous to mitosis during cell-to-cell movement of invasive hyphae. These results provide new insight into the potential roles of the appressorium after host penetration and highlight the unique mitotic dynamics during rice blast infection.

Cell Biology↗

bioRxiv2016

Nuclear and structural dynamics during the establishment of a specialized effector-secreting cell by Magnaporthe oryzae in living rice cells

BackgroundTo cause an economically important blast disease on rice, the filamentous fungus Magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. Once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae (IH), which are surrounded by a host-derived membrane. These hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex (BIC). The first IH cell, also known as the side BIC-associated cell, is a specialized effector-secreting cell essential for a successful infection. This study aims to determine cellular processes that lead to the development of this effector-secreting first IH cell inside susceptible rice cells.\n\nResultsUsing live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first IH cell in live rice cells. The filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. The single nucleus in the appressorium divided, and then one nucleus migrated into the swollen apex. Septation occurred in the filamentous region of the primary hypha, establishing the first IH cell. The tip BIC that was initially associated with the primary hypha becomes the side BIC on the swollen apex prior to nuclear division in the appressorium. The average distance between the early side BIC and the nearest nucleus in the appressorium was estimated to be more than 32 {micro}m. These results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion to the distantly located BIC. When M. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous IH cell, suggesting its specialization for establishment of biotrophic infection.\n\nConclusionsOur studies reveal cellular dynamics associated with the development of the effector-secreting first IH cell. Our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the BIC.

Cell Biology↗

bioRxiv2016

Actomyosin Contractility Drives Bile Regurgitation as an Early Homeostatic Response to Increased Biliary Pressure in Obstructive Cholestasis

A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response to abnormal elevations in biliary pressure remains poorly elucidated. Bile canaliculi are dynamic luminal structures that undergo actomyosin-mediated periodic contractions to propel secreted bile. Additionally, pericanalicular actin is accumulated during obstructive cholestasis. Therefore, we hypothesize that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response against increased biliary pressure. Here, we report that, actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex, and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8 {micro}m). Importantly, we show that these vesicles aid in the regurgitation of bile from the canalicular system. Conclusion: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis.

Cell Biology↗

bioRxiv2016

Interaction between distinct pools controls activity-dependent actin dynamics in the dendritic spine

Actin cytoskeleton is composed of functionally distinct pools of filamentous (F)-actin defined by their regulatory machinery and dynamics. Although these networks may compete for actin monomers and regulatory factors1-4, the interaction between them remains poorly understood. Here, we show that disruption of the labile F-actin pool in neurons by limited actin depolymerization5,6 unexpectedly triggers rapid enhancement of the F-actin content at the dendritic spine. Long-term blockade of NMDA-type receptors decreases spine actin polymerization, which is specifically restored by the labile pool ablation. Increase in the spine actin is triggered by blockade of formin-induced actin polymerization in a manner dependent on Arp2/3 complex activity. Finally, limited actin depolymerization increases F-actin levels in a cultured cell line, suggesting the generality of the two-tiered actin dynamics. Based on these findings, we propose a model whereby the labile pool of F-actin controlled by formin restricts the polymerization state of the Arp2/3-regulated stable spine actin, suggesting a feedback principle at the core of cytoskeletal organization in neurons.\n\nHighlightsO_LIDisruption of labile F-actin by limited depolymerization rapidly increases the synaptic F-actin content;\nC_LIO_LIThe depolymerization-induced F-actin boost reverses decrease in synaptic F-actin induced by long-term NMDA receptor blockade;\nC_LIO_LIBlockade of formin-dependent actin polymerization boosts synaptic F-actin in an Arp2/3-dependent manner;\nC_LIO_LILimited actin depolymerization enhances overall F-actin content in a mammalian cell line.\nC_LI

Cell Biology↗

bioRxiv2016

Heterogeneity of single-cell mechanical responses to tumorigenic factors.

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction followed by cell de-adhesion, scattering and invasion. Tumorigenic factors may act specifically on one of these steps or have wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type-I/fibronectin on deformable surfaces to standardize cell behavior and to measure simultaneously cell size, speed of motion and the magnitude of the associated contractile forces at the level of a single cell. We analyzed and compared normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, distinct populations of cells with a wide range of biomechanical properties were identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or the loss of the beta subunit of casein kinase 2 (CK2), shifted the whole population towards a faster speed and lower contractility state. Treatment with transforming growth factor beta (TGF-{beta}), induced some cells to adopt opposing behaviors such as extreme high contractility versus extreme low contractility. Thus, tumor transformation amplified the pre-existing population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than that observed with normal cells.

Cell Biology↗

bioRxiv2016

Protein Kinase C and the stress response pathways are required for kinetochore homeostasis.

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. To identify proteins that regulate the kinetochore we used a genome-wide fluorescence microscopy approach. We combined an array of mutants of either non-essential gene deletions or essential temperature-sensitive alleles with fluorescently tagged spindle pole bodies (centrosome) and outer kinetochores. Quantitative and qualitative analysis revealed mutants that affect the levels and distribution of kinetochores respectively. These mutants are enriched for those involved in mRNA processing, chromatin organization, DNA replication/repair and mitosis. Our data show that the Pkc1 kinase maintains the kinetochore focus via its ability to prevent cell stress and this phenotype is rescued by an osmotic stabilizer. These data support the notion that kinetochore and microtubule homeostasis are perturbed by the stress response pathways. Hence this observation provides a candidate mechanism for extracellular stress leading to chromosome segregation defects.

Cell Biology↗

bioRxiv2016

Myosin II controls junction fluctuations to guide epithelial tissue ordering

Homophilic interactions between E-Cadherin molecules generate adhesive interfaces or junctions (AJs) that connect neighbouring cells in epithelial monolayers. These are highly dynamic structures. Under conditions of homeostasis, changes in the length of individual interfaces provide epithelia with the fluidity required to maintain tissue integrity in the face of cell division, delamination and extrinsic forces. Furthermore, when acted upon by polarized actomyosin-based forces, changes in AJ length can also drive neighbour exchange to reshape an entire tissue. Whilst the contribution of AJ remodelling to developmental morphogenesis has been subjected to intensive study, less is known about AJ dynamics in other circumstances. Here, using a combination of experiment and computational modelling, we study AJ dynamics in an epithelium that undergoes a gradual increase in packing order without concomitant large-scale changes in tissue shape or size. Under these conditions, we find that neighbour exchange events are driven by stochastic fluctuations in junction length, which are regulated at least in part by the level of junctional actomyosin. As a result of this behaviour, the steady increase in junctional actomyosin and consequent tension that accompanies development steadily reduces the rate of neighbour exchange and orders the tissue. This leads us to propose a model in which topological transitions, that underpin tissue fluidity, are either inhibited or biased by actomyosin-based forces, to drive, respectively, tissue ordering or deformation.

Cell Biology↗

bioRxiv2016

Size-Dependent Accumulation of the Mitotic Activator Cdc25 as a Mechanism of Size Control in Fission Yeast

Proper cell size is essential for cellular function (Hall et al., 2004). Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell size homeostasis are largely mysterious (Marshall et al., 2012). Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range, and returns the population average to that range if it is perturbed (Marshall et al., 2012; Turner et al., 2012; Amodeo and Skotheim, 2015). Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition (Schmoller et al., 2015), while bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average, a mechanism also suggested for budding yeast (Campos et al., 2014; Jun and Taheri-Araghi, 2015; Taheri-Araghi et al., 2015; Tanouchi et al., 2015; Soifer et al., 2016). Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size dependent expression of the mitotic activator Cdc25. The cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cell grow and providing a mechanism for cell to trigger cell division when they reach a threshold concentration of Cdc25. Since regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a wide spread solution to the problem of size control in eukaryotes.

Cell Biology↗

bioRxiv2016

Wound induced Ca2+ wave propagates through a simple Release and Diffusion mechanism

Damage associated molecular patterns (DAMPs) are critical mediators of information concerning tissue damage from damaged cells to neighboring healthy cells. Adenosine triphosphate (ATP) acts as an effective DAMP when released into extracellular space from damaged cells. Extracellular ATP receptors monitor tissue damage and activate a Ca2+ wave in the surrounding healthy cells. How the Ca2+ wave propagates through cells following a wound is unclear. Ca2+ wave activation can occur extracellularly via external receptors or intracellularly through GAP junctions. Three potential mechanisms to propagate the Ca2+ wave are: Source and Sink, Amplifying Wave, and Release and Diffusion. Both Source and Sink and Amplifying Wave regulate ATP levels using hydrolysis or secretion, respectively, while Release and Diffusion relies on dilution. Here we systematically test these hypotheses using a microfluidics assay to mechanically wound an epithelial monolayer in combination with direct manipulation of ATP hydrolysis and release. We show that a Release and Diffusion model sufficiently explains Ca2+ wave propagation following an epithelial wound. A Release and Diffusion model combines the benefits of fast activation at length-scales of ~1-5 cell diameters with a self-limiting response to prevent unnecessary inflammatory responses harmful to the organism.

Cell Biology↗

bioRxiv2016

Microtubule stabilization drives centrosome migration to initiate primary ciliogenesis

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface. Yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetric contraction participated in concert to destabilize basal centrosome position and drive apical centrosome migration. The distal appendage protein Cep164 appeared to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas IFT88's role seemed to be restricted to axoneme elongation. Together our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.

Cell Biology↗

bioRxiv2016

The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast

The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast.

Cell Biology↗

bioRxiv2016

The p150N domain of Chromatin Assembly Factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer

Chromatin Assembly Factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but which promotes the localization of specific loci (Nucleolar-Associated Domains, or \"NADs\") and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis, and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the Sumoylation Interacting Motif (SIM) within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.

cell biology↗

bioRxiv2016

Casein Kinase II is Required for Proper Cell Division and Acts as a Negative Regulator of Centrosome Duplication in C. elegans Embryos

Summary statementThe conserved protein kinase CK2 negatively regulates centrosome assembly and is required for proper cell cycle progression and cytokinesis in early C. elegans embryos.\n\nAbstractCentrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein Kinase II (CK2) in early C. elegans embryos. The catalytic subunit (KIN-3/CK2) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

cell biology↗

bioRxiv2016

CENP-F couples cargo to growing and shortening microtubule ends

Dynamic microtubule ends exert pulling and pushing forces on intracellular membranes and organelles. However, the mechanical linkage of microtubule tips to their cargoes is poorly understood. CENP-F is a non-motor microtubule-binding protein that participates in microtubule binding at kinetochores and in the mitotic redistribution of the mitochondrial network. CENP-F-driven mitochondrial transport is linked to growing microtubule tips, but the underlying molecular mechanisms are unknown. Here we show that CENP-F tracks growing microtubule ends in living cells. In vitro reconstitution demonstrates that microtubule tips can transport mitochondria and CENP-F-coated artificial cargoes over micrometer-long distances, during both growing and shrinking phases. Based on these and previous observations, we suggest that CENP-F might act as a transporter of mitochondria and other cellular cargoes by attaching them to dynamic microtubule ends.

cell biology↗