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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

CSB-dependent CDK9 degradation and RNA Polymerase II phosphorylation during Transcription Coupled Repair

AUTHOR SUMMARYDNA lesions block cellular processes such as transcription, inducing apoptosis, tissue failures and premature ageing. To counteract the deleterious effects of DNA damage, cells are equipped with various DNA repair pathways. Transcription Coupled Repair specifically removes helix-distorting DNA adducts in a coordinated multi-step process. This process has been extensively studied, however once the repair reaction is accomplished, little is known about how transcription restarts. In this study, we show that, after UV irradiation, the CDK9/CyclinT1 kinase unit is specifically released from the HEXIM1 complex and that this released fraction is degraded in the absence of CSB. We determine that UV-irradiation induces a specific Ser2 phosphorylation of the RNA polymerase II and that this phosphorylation is CSB dependent. Surprisingly CDK9 is not responsible for this phosphorylation but instead plays a non-enzymatic role in transcription restart after DNA repair.

cell biology↗

bioRxiv2018

PID1 alters antilipolytic action of insulin and increases lipolysis via Inhibited the activation of AKT/PKA Pathway

PurposeThe aim was to investigate the mechanism for impaired control of lipolysis in obesity by investigating the effect of PID1 on insulin-induced activation of AKT/PKA/HSL pathway and lipolysis.\n\nMethodsFirst, PID1 expression was detected in adipose tissue and blood insulin and glycerol levels were measured in high-fat diet-induced obese rats. Next, we examined the effect of different concentrations of insulin on lipolysis and AKT/PKA/HSL pathway in 3T3-L1cells. We also investigated the role of PID1 in regulating AKT/PKA/HSL cascade and lipolysis after insulin treatment and lipofectamine over-expression.\n\nResultsPID1 expression is increased in adipose tissue from HFD rat and positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that antilipolytic effect of insulin is mediated by AKT and AKT activated by insulin can results in phosphorylation of PKA and HSL and suppresses glycerol release. However, over-expression of PID1 counteracts insulin action as indicated by glycerol releaseand reduced level of Akt phosphorylation in accordance with a decrease in the activity of insulin-dependent PKA/HSLsignaling cascade.\n\nConclusionsAll together, these data showed that activation of PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may constitute a new strategy to ameliorate adipocyte lipolysis and hence to improve insulin sensitivity.

cell biology↗

bioRxiv2018

PLK1 plays dual roles in centralspindlin regulation during cytokinesis

Cytokinesis starts in anaphase with the formation of an actomyosin-based contractile ring at the equatorial cortex, which is governed by the local activation of the small GTPase RhoA. Here we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin oligomerization at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone making it unavailable for Aurora B-dependent oligomerization at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.

cell biology↗

bioRxiv2018

Chronic starvation induces non-canonical pro-death stress granules

Stress granules (SGs) assemble under stress-induced conditions that inhibit protein synthesis, including eIF2 phosphorylation, inhibition of the RNA helicase eIF4a, or inactivation of mTORC1. Classically defined SGs are composed of translation initiation factors, 40S ribosomes, RNA binding proteins, and poly(A)+ mRNAs, and as such represent an important compartment for storage of mRNAs and regulation of their translation. Emerging work on SGs indicates they may play important roles in cancer, neurodegenerative disease, and viral infection, often promoting survival. Yet much previous work on SGs formation and function has employed acute stress conditions, which may not accurately reflect the chronic stresses that manifest in human disease. We used prolonged nutrient starvation to investigate SG formation and function during chronic stress. Surprisingly, SGs that form under chronic nutrient starvation lack 40S ribosomes, do not actively exchange their constituent components with cytoplasmic pools, and promote cell death. These results imply that SG assembly and function in the context of prolonged nutrient starvation stress differ significantly from what has been described for acute stress conditions.\n\nSummary StatementThis work characterizes the mechanisms of formation of a novel type of stress granule that is induced in response to long-term starvation, and unlike previously described stress granules, functions in a pro-death capacity.

cell biology↗

bioRxiv2018

Optogenetic dissection of Rac1 and Cdc42 gradient shaping

During migration, cells present a polarized activity that is aligned with the direction of motion. This cell polarity is established by an internal molecular circuitry, without the requirement of extracellular cues. At the heart of this circuitry, Rho GTPases spontaneously form spatial gradients that define the front and back of migrating cells. At the front of the cell, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away from the cell tip. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Combining optogenetics and cell micopatterning, we show that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by advection or diffusion mechanisms. Cdc42 simply follows the distribution of GEFs thanks to a uniform GAP activity, whereas Rac1 shaping requires the activity of an additional GAP, {beta}2-chimaerin, which is sharply localized at the tip of the cell. We find that {beta}2-chimaerin recruitment depends on feedbacks from Cdc42 and Rac1. Functionally, the extent -neither the slope nor the amplitude- of RhoGTPases gradients governs cell migration. A Cdc42 gradient with a short spatial extent is required to maximize directionality during cell migration while an extended Rac1 gradient controls the speed of the cell.

cell biology↗

bioRxiv2018

Actin cytoskeleton self-organization in single epithelial cells and fibroblasts under isotropic confinement

We systematically investigated the principles of actin cytoskeleton self-organization in two cell types, fibroblasts and epitheliocytes, by confining isolated cells on isotropic adhesive islands of varying size. In fibroblasts, we previously described that an initially circular pattern of circumferential actin dynamically evolves into a radial pattern of actin bundles that spontaneously transforms into a chiral pattern, before finally producing parallel linear stress fibres. We now show that progression from circular to chiral actin patterns depends on cell projected area and rarely occurs on small islands. Epitheliocytes however, did not exhibit succession through all the actin patterns described above even on large islands. Upon confinement, the actin cytoskeleton in non-keratinocyte epitheliocytes is arrested at the circular stage, while in keratinocytes it can progress as far as the radial pattern but still cannot break symmetry. Epithelial-mesenchymal transition pushed actin cytoskeleton development from circular towards radial patterns but remains insufficient to cause chirality. Surprisingly, small doses of G-actin sequestering drug, latrunculin A, induced chiral swirling in keratinocytes. During this swirling, keratin filaments follow actin and also demonstrate chiral swirling movement. Elimination of the keratin network by genetic silencing of Type II keratins, however, did not affect the self-organization of the actin cytoskeleton.

cell biology↗

bioRxiv2018

Variants of DNMT3A cause transcript-specific DNA methylation patterns and affect hematopoietic differentiation

The de novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing of DNMT3A has characteristic epigenetic and functional sequels. Specific DNMT3A transcripts were either downregulated or overexpressed in human hematopoietic stem and progenitor cells and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that particularly transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia (AML) expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating that DNMT3A variants impact also on malignancies. Our results demonstrate that specific DNMT3A variants have distinct epigenetic and functional impact. Particularly DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in AML.

cell biology↗

bioRxiv2018

Long-term expanding human airway organoids for disease modelling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar biopsies or lavage material. The pseudostratified airway organoid epithelium consists of basal cells, functional multi-ciliated cells, mucus-producing goblet cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoid culture conditions also allow gene editing as well as the derivation of various types of lung cancer organoids. Respiratory syncytial virus (RSV) infection recapitulated central disease features and dramatically increases organoid cell motility, found to be driven by the non-structural viral NS2 protein. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

cell biology↗

bioRxiv2018

Local Contractions Test Rigidity of E-Cadherin Adhesions

E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Since epithelial cells respond to rigidity and tension in the tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using sub-micrometer, E-cadherin-coated PDMS pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require non-muscle myosin IIB, tropomyosin 2.1, -catenin and binding of vinculin to -catenin; plus, they are correlated with rigidity-dependent cell spreading. Without contractions, cells fail to spread to different areas on soft and rigid surfaces and to maintain monolayer integrity. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells, and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions.

cell biology↗

bioRxiv2018

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-{kappa}B activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-{kappa}B at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

cell biology↗

bioRxiv2018

Basal level of p53 regulates cell population homeostasis

The tumor suppressor p53 regulates various stress responses via increasing its cellular levels. The lowest p53 levels occur in unstressed cells; however, the functions of these low levels remain unclear. To investigate the functions, we used empirical single-cell tracking of p53-expressing (Control) cells and cells in which p53 expression was silenced by RNA interference (p53 RNAi). Here we show that p53 RNAi cells underwent more frequent cell death and cell fusion, which further induced multipolar cell division to generate aneuploid progeny. Those results suggest that the low levels of p53 in unstressed cells indeed have a role in suppressing the induction of cell death and the formation of aneuploid cells. We further investigated the impact of p53 silencing by developing an algorithm to simulate the fates of individual cells. Simulation of the fate of aneuploid cells revealed that these cells could propagate to create an aneuploid cell population. In addition, the simulation also revealed that more frequent induction of cell death in p53 RNAi cells under unstressed conditions conferred a disadvantage in terms of population expansion compared with Control cells, resulting in faster expansion of Control cells compared with p53 RNAi cells, leading to Control cells predominating in mixed cell populations. In contrast, the expansion of Control cells, but not p53 RNAi cells, was suppressed when the damage response was induced, allowing p53 RNAi cells to expand their population compared with the Control cells. These results suggest that, although p53 could suppress the formation of aneuploid cells, which could have a role in tumorigenesis, it could also allow the expansion of cells lacking p53 expression when the damage response is induced. p53 may thus play a role in both the suppression and the promotion of malignant cell formation during tumorigenesis.

cell biology↗

bioRxiv2018

Cell type-dependent effects of ellagic acid on cellular metabolism

Ellagic acid is a botanical polyphenol which has been shown to have numerous effects on cellular function. Ellagic acid can induce apoptosis and inhibit the proliferation of various cancer cell types in vitro and in vivo. As such, ellagic acid has attracted significant interest as a potential chemotherapeutic compound. One mechanism by which ellagic acid has been proposed to affect cellular physiology is by regulating metabolic pathways. Here we show the dose-dependent effects of ellagic acid on cellular energy production and downstream induction of the apoptotic program in HEK293, HeLa, MCF7, and HepG2 cells. At physiologically relevant doses, eallgic acid has pleiotropic and cell-type specific effects on mitochondrial function. At high doses ellagic acid can also influence glycolytic pathways and induce cell death. Our results demonstrate that ellagic acid can influence mitochondrial function at therapeutically relevant concentrations. The observed effects of ellagic acid on cellular respiration are complex and cell type-specific, which may limit the chemotherapeutic utility of this compound.

cell biology↗

bioRxiv2018

Programmed Variations of Cytokinesis Contribute to Morphogenesis in the C. elegans embryo

While cytokinesis has been intensely studied, how it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant C. elegans embryo lineage and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we find several unexpected alterations including migration of midbodies to the apical surface during epithelial polarization in different tissues. Aurora B kinase, which is essential for several aspects of cytokinesis, remains localized to the apical membrane after internalization of other midbody components. Inactivation of Aurora B causes cytokinesis failure, which disrupts polarization and tissue formation. Therefore, cytokinesis shows surprising diversity during development and is required during epithelial polarization to establish cellular architecture during morphogenesis.

cell biology↗

bioRxiv2018

Optogenetic dissection of mitotic spindle positioning in vivo

The position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of G-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through G-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the G regulators RIC-8Ric-8A and RGS-7Loco/RGS3, and replacement of G with a light-inducible membrane anchor demonstrated that G-GTP signaling is dispensable for pulling force generation. In the absence of G, cortical recruitment of GPR-1/2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment of LIN-5 overruled normal cell-cycle and polarity regulation, and provided experimental control over the spindle and cell cleavage plane. Our results define G*GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle positioning forces. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.

cell biology↗

bioRxiv2018

Brief Freezing Steps Lead to Robust Immunofluorescence in Drosophila Larval Brains

Drosophila melanogaster possess complex neuronal networks regulating sophisticated behavioral outputs that aid in studying the molecular mechanisms of neuronal function and neurodegenerative disease. Immunofluorescence (IF) techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling, and eventually - degradation. However, general immunostaining techniques do not always result in sufficient antibody penetration through the brain, and techniques used to enhance permeability can compromise structural integrity. We have found that freezing larval brains facilitates permeability with no apparent loss of antibody specificity or structural integrity. To demonstrate the advantage of this freezing technique, we compared results to two commonly used permeation methods: Detergent alone (Basic) and proteolytic degradation (Collagenase) techniques.\n\nSummaryHere we compare four different immunofluorescence techniques demonstrating that freezing Drosophila brains results in robust staining of small neurons in the larval brain without compromising structural integrity.

cell biology↗

bioRxiv2018

Automating multimodal microscopy with NanoJ-Fluidics

Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary information. Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows involving multiple sample preparation steps. We present a robust fluidics approach to automate complex sequences of treatment, labelling and imaging of live and fixed cells. Our open-source NanoJ-Fluidics system is based on low-cost LEGO hardware controlled by ImageJ-based software and can be directly adapted to any microscope, providing easy-to-implement high-content, multimodal imaging with high reproducibility. We demonstrate its capacity to carry out complex sequences of experiments such as super-resolved live-to-fixed imaging to study actin dynamics; highly-multiplexed STORM and DNA-PAINT acquisitions of multiple targets; and event-driven fixation microscopy to study the role of adhesion contacts in mitosis.

cell biology↗

bioRxiv2018

Tumor suppressor DAPK1 catalyzes adhesion assembly on rigid but anoikis on soft matrices

Cancer cells will normally grow on soft surfaces, but if rigidity sensing modules are restored in cancer cells, they will undergo apoptosis on soft surfaces (anoikis) like most normal cells. DAPK1 is a major tumor suppressor that activates cell death, but it is unclear how DAPK1 could activate anoikis through rigidity sensing. Here we find that when rigidity sensing is decreased through inhibition of DAPK1 activity, cells are transformed for growth on soft matrices. Further, DAPK1 catalyzes matrix adhesion assembly and is part of adhesions on rigid surfaces. Additional factors include DAPK1 phosphorylation of tropomyosin2.1, talin1 head domain and tyrosine phosphorylation of DAPK1 by Src. On soft surfaces, DAPK1 rapidly dissociates from the adhesion complexes and activates apoptosis that requires PTPN12 activity and talin1 head. Thus, DAPK1 is important for adhesion assembly on rigid surfaces and the activation of anoikis on soft surfaces through its binding to rigidity-sensing modules.

cell biology↗

bioRxiv2018

PP2A-Rts1 enforces a proportional relationship between cell size and growth rate

Cell size is proportional to growth rate. Thus, cells growing slowly in poor nutrients can be nearly half the size of cells growing rapidly in rich nutrients. The relationship between cell size and growth rate appears to hold across all orders of life, yet the underlying mechanisms are unknown. In budding yeast, most growth occurs during mitosis, and the proportional relationship between cell size and growth rate is therefore enforced primarily by modulating growth in mitosis. When growth is slow, the duration of mitosis is increased to allow more time for growth, yet the amount of growth required to complete mitosis is reduced, leading to birth of small daughter cells. Previous studies found that PP2A associated with the Rts1 regulatory subunit (PP2ARts1) works in a TORC2-dependent feedback loop that sets cell size and growth rate to match nutrient availability. However, it was unknown whether PP2ARts1 influences growth in mitosis. Here, we show that PP2ARts1 is required for the proportional relationship between cell size and growth rate during mitosis. Moreover, nutrients and PP2ARts1 influence the duration of mitosis, and thus the extent of growth in mitosis, via Wee1 and Pds1/securin, two conserved regulators of mitotic progression. Together, the data suggest a model in which the same global signals that set growth rate also set the critical amount of growth required for cell cycle progression, which would provide a simple mechanistic explanation for the proportional relationship between size and growth rate.

cell biology↗