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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,315 records · Page 73Linked to original sources

bioRxiv2018

GGA2 and RAB13 regulate activity-dependent β1-integrin recycling

{beta}1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes like cancer cell invasion. Here we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with {beta}1-integrin and its role in recycling of the active but not inactive {beta}1-integrin receptors. Silencing of GGA2 limits active {beta}1-integrin levels in focal adhesions and decreases cancer cell migration and invasion congruent with its ability to regulate the dynamics of active integrins. Using the proximity-dependent biotin identification (BioID) method, we identify two RAB family small GTPases, RAB13 and RAB10, associating with GGA2 and {beta}1-integrin. Functionally, RAB13 silencing triggers the intracellular accumulation of active {beta}1-integrin, reduces integrin activity, in focal adhesions, and cell migration, similarly to GGA2 depletion, indicating that both facilitate active {beta}1-integrin recycling the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.

cell biology↗

bioRxiv2018

Nicastrin haploinsufficiency alters expression of type-I interferon-stimulated genes in two immortalized human cell lines

A.BackgroundHidradenitis suppurativa (HS) is a chronic skin disease. The symptoms can be severe, and include intensely painful nodules and abscesses in apocrine-gland rich inverse skin, such as the buttocks, under the arms, and the groin. Autosomal dominant forms of HS exist, but are rare. Some of these kindred have heterozygous loss-of-function rare variants in the {gamma}-secretase complex component nicastrin (NCSTN).\n\nObjectivesWe wanted to know what effect NCSTN haploinsufficiency has on human keratinocytes to assess potential mechanisms for lesion development.\n\nMethodsWe knocked down nicastrin using an shRNA construct in both a keratinocyte cell line (HEK001) and an embryonic kidney cell line (HEK293). We assessed differential gene expression using RNA microarray. We also generated a NCSTN heterozygous deletion in the HEK293 line using CRISPR/Cas9 genome-editing and assessed NFKB activity in this line using a luciferase reporter.\n\nResultsThe keratinocyte NCSTN knockdown cell line demonstrated significantly increased expression of genes related to the type-I interferon response pathway when compared to controls. Both HEK001 and HEK293 knockdowns demonstrated evidence for altered growth. We observed a small, but significant increase in NFKB signaling in response to TNF treatment a HEK293 line genome-edited for reduced NCSTN.\n\nConclusionsOur data suggest a role for increased keratinocyte inflammatory responsiveness in familial HS. Confirming this phenotype, and characterizing additional effects in different cell types, will require study beyond cell lines in primary cells and tissues.

cell biology↗

bioRxiv2018

Yeast replicative aging leads to permanent cell cycle arrest in G1 effectuated by the start repressor Whi5

Yeast replicative aging has been a canonical model for aging research. Since replicative aging eventually leads to permanent cell cycle arrest, a fundamental question is how cells sense the signals from aging and communicate that to the cell cycle control machineries. Using microfluidic devices to track individual mother cells labeled by two different cell cycle markers Whi5-tdTomato and Myo1-EGFP, we measured the length of different cell cycle phases as a function of age and the distribution of cell death in different cell cycle phases. We found that the majority of the cells died in the G1 phase, and their G1 cell cycle length increased drastically in the last few cell divisions. This increase of G1 length correlates with the increase of the nuclear concentration of Whi5, which is a major transcriptional suppressor of the cell cycle start check point. Interestingly, this correlation is apparent only above a threshold concentration of Whi5. We show that in response to external stress, Whi5 concentration increases and cell growth slows down in a Whi5 dependent manner, and that Whi5 deletion significantly extends the lifespan. Together these data suggest the existence of a programmed control to arrest cell cycle in G1 in response to stress signals due to aging, and that Whi5 is a major mediator of this process. Our findings may have important implications in understanding senescence and cancer in mammalian cells, which have a parallel G1/S control system with Rb (a well known tumor suppressor) as the analog of Whi5.\n\nSignificance statementIn this work, we used microfluidic devices to track individual mother cells labeled by two cell cycle markers Whi5-tdTomato and Myo1-EGFP. We found that aging leads to significant lengthening of G1 phase in old cells and the eventual permanent cell cycle arrest in G1, and Whi5 plays an important role in implementing such a program. We show that oxidative stress can lead to the increase of Whi5 expression and the slow-down of cell division. Furthermore, Whi5 deletion significantly extends the lifespan. The result suggest the existence of a programmed control to arrest cell cycle in G1 in response to stress signals due to aging, and that Whi5 is a major mediator of this process.

cell biology↗

bioRxiv2018

Growth differentiation factor-15 regulates oxLDL-induced lipid homeostasis and autophagy in human macrophages

Growth differentiation factor-15 (GDF-15), a divergent and distant member of the transforming growth factor-{beta} superfamily, is suggested as a risk factor for cardiovascular diseases. Thus, we are interested to investigate the influence of GDF-15 in lipid homeostasis and autophagy in macrophages (M{Phi}) during foam cell formation. Our investigations represent the impairment of GDF-15 on modulators of autophagy and lipid homeostasis in PMA-differentiated human THP-1 M{Phi}. In this context, in vitro resulted GDF-15 silencing in a reduction of lipid accumulation, whereas the addition of recombinant (r)GDF-15 increased the lipid accumulation in human M{Phi} independent of oxidized (ox)LDL. Additionally, GDF-15 affected the expression of autophagy-relevant proteins (p62, Atg5 and Atg12/Atg5 protein complex) and the p62 accumulation in THP-1 M{Phi}. Hence, our data suggest that GDF-15 is involved in the regulation of the lipid homoeostasis of human M{Phi} by regulating autophagic processes.

cell biology↗

bioRxiv2018

Identification of STK25 as a direct activator of LATS signaling

The Hippo pathway maintains tissue homeostasis by negatively regulating the oncogenic transcriptional co-activators YAP and TAZ. Though functional inactivation of the Hippo pathway is common in tumors, mutations in core pathway components are rare. Thus, understanding how tumor cells inactivate Hippo signaling remains a key unresolved question. Here, we identify the kinase STK25 as a novel activator of Hippo signaling. We demonstrate that loss of STK25 promotes YAP/TAZ activation and enhanced cellular proliferation, even under normally growth-suppressive conditions. We reveal that STK25 activates LATS via a previously unobserved mechanism, in which STK25 directly phosphorylates the LATS activation loop. This represents a new paradigm in Hippo activation and distinguishes STK25 from all other identified kinase activators of LATS. STK25 is significantly focally deleted across a wide spectrum of human cancers, suggesting STK25 loss may represent a common mechanism by which tumor cells functionally impair the Hippo tumor suppressor pathway.

cell biology↗

bioRxiv2018

Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

cell biology↗

bioRxiv2018

Exocyst Dynamics During Vesicle Tethering and Fusion

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of Sec3 occurs prior to fusion, whereas other subunits depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.

cell biology↗

bioRxiv2018

A new calcium-activated dynein adaptor protein, CRACR2a, regulates clathrin-independent endocytic traffic in T cells

Cytoplasmic dynein is a microtubule minus-end-directed motor that transports numerous intracellular cargoes. Mammalian dynein transport is initiated by coiled-coil adaptor proteins that 1) join dynein and its co-factor dynactin into a complex capable of processive motility, and 2) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, which have a coiled-coil adaptor domain, a pair of EF hands, and a Rab GTPase domain fused into a single polypeptide. We find that CRACR2a-mediated dynein-dynactin motility is activated by calcium in vitro and in cells. In activated T cells, CRACR2a localizes to clathrin-independent endosomes that require microtubule-based transport to detach from the actin cortex and travel towards the microtubule organizing center. Together these results represent the first known examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in regulation of endocytic trafficking in T cells.

cell biology↗

bioRxiv2018

ATR repression at telomeres by POT1a and POT1b: RPA exclusion and interference by CST

Telomeres carry a constitutive 3 overhang that can bind RPA and activate ATR signaling. POT1a, a single-stranded (ss) DNA binding protein in mouse shelterin, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires the TPP1/TIN2 mediated tethering of POT1a to the the rest of the shelterin complex situated on the ds telomeric DNA. The simplest version of the tethered exclusion model for ATR repression suggests that the only critical features of POT1a are its connection to shelterin and its binding to ss telomeric DNA binding. In agreement with the model, we show that a shelterin-tethered RPA70 mutant, lacking the ATR recruitment domain, is effective in repressing ATR signaling at telomeres. However, arguing against the simple tethered exclusion model, the nearly identical POT1b subunit of shelterin is much less proficient in ATR repression than POT1a. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to the CST/Pol/primase complex. The data establish that shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the 3 overhang and suggest that ATR repression does not require the interaction of POT1 with the 3 end or G4 DNA.

cell biology↗

bioRxiv2018

ZO-2 induces cytoplasmic retention of YAP by promoting a LATS1-ZO-2-YAP complex at tight junctions

Contact inhibition of proliferation (CIP) is a key mechanism that transduces the cell density status of tissue and organs into a unique transcriptional program by translocating YAP between the nucleus and the cytoplasm. However, the nature of the cell density-dependent cues that regulate the YAP distribution remains unclear. Here, we present evidence that tight junctions serve as a platform that controls both distribution and activity of LATS1, a kinase that phosphorylates YAP. This CIP effect is mediated by the scaffold function of junctional protein, ZO-2, by promoting LATS1 interaction with YAP in the cytoplasm, and then targeting the tripartite complex to tight junctions. There, LATS1 is activated by angiomotin and NF2, thereby stimulating YAP phosphorylation and its cytoplasmic retention. Our findings delineate novel mechanisms governing CIP, in which ZO-2 utilizes the status of cell-cell cohesion to control the phosphorylation status and therefore inactivation of YAP by LATS1 in the cytoplasm.

cell biology↗

bioRxiv2018

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.

cell biology↗

bioRxiv2018

CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization

Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18, Mis18{beta}, M18BP1). Here we show that Xenopus M18BP1 localizes to centromeres during metaphase - prior to CENP-A assembly - by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase.

cell biology↗

bioRxiv2018

A 14-day Dexamethasone timecourse exposure in Caco-2 monolayers results in differential expression of tight-junction and cytoskeleton regulatory pathway genes

Glucocorticoid (GC) hormones are modulators of endogenous stress responses and are important pharmaceuticals for inflammatory and autoimmune diseases. The gastrointestinal epithelium is a significant tissue target of Glucocorticoids; perturbation of epithelial barrier function during the endogenous stress response plays a major role in the pathophysiology of inflammatory bowel disease. Epithelial permeability and barrier function are mediated by the tight junction protein complex, with a network of molecular/cellular interactions occurring between the actin cytoskeleton, RhoGTPase, Akt/PI3K and growth factor receptor, and inflammatory cytokine signaling, influencing the establishment of a partial EMT phenotype. To improve our understanding of GC-responsive gene expression in a gastrointestinal epithelial context, we tested polarized Caco-2 monolayer cultures during at 30-day timecourse, with 15-days of continuous Dexamethasone exposure. Trans-epithelial resistance (TEER) was recorded to provide a physiological quantification of barrier function during the timecourse treatments. Presence of intracellular glucocorticoid hormone activates the human glucocorticoid receptor (GCR, human NR3C1 gene) transcription factor, resulting in transcriptional activation and repression of various GCR-responsive genes. We tested for differential gene expression with a multiplexed panel of 250 gene expression panel using the Nanostring nCounter(R) system. Gene panel selection was based on membership of genes in canonical KEGG pathways for tight-junction, adherens junction, focal adhesion, actin cytoskeleton regulation, and colorectal cancer. Our TEER results confirm, as previously reported, that long-term Dexamethasone exposure results in decreased permeability in Caco-2 monolayers ~day 20-25. Culture age and Dexamethasone exposure both contributed to differential gene expression for cell-cell junction, protein kinases, survival and cancer associated genes, reported here in the context of their corresponding KEGG pathway representations. The findings give evidence for GC and time-associated patterns of transcriptional response and provide further insight into long-term glucocorticoid-associated physiological effects on the gastrointestinal epithelia. Future research utilizing more advanced cell culture methods will use this data-set as a reference.

cell biology↗

bioRxiv2018

A somatic evolutionary model of the dynamics of aneuploid cells during hematopoietic reconstitution

Aneuploidy is associated with many cancers. Recent studies demonstrate that in thehematopoietic stem and progenitor cell (HSPC) compartment aneuploid cells havereduced fitness and are efficiently purged from the bone marrow. However, early phasesof hematopoietic reconstitution following bone marrow transplantation provide awindow of opportunity whereby aneuploid cells rise in frequency, only to decline to basallevels thereafter. Here we demonstrate by Monte Carlo modeling that two mechanismscould underlie this aneuploidy peak: rapid expansion of the engrafted HSPC populationand bone marrow microenvironment degradation caused by pre-transplantationradiation treatment. Both mechanisms reduce the strength of purifying selection actingin early post-transplantation bone marrow. We explore the contribution of other factorssuch as alterations in cell division rates that affect the strength of purifying selection, thebalance of drift and selection imposed by the HSPC population size, and the mutationselectionbalance dependent on the rate of aneuploidy generation per cell division. Wepropose a somatic evolutionary model for the dynamics of cells with aneuploidy or otherfitness-reducing mutations during hematopoietic reconstitution following bone marrowtransplantation.\n\nSignificanceBone marrow transplantations (BMT) following ablative irradiation pose a great health risk. Its been shown that additionally the bone microenvironment is conducive to elevated frequencies of aneuploid cells in mice during bone marrow reconstitution post-BMT. As aneuploidy is linked with many cancers, we explore the reasons of such aberrant cell frequency peaks by Monte Carlo modeling. We demonstrate that elevated rates of aneuploidy early post-BMT are likely to be caused by reduced purifying somatic selection resulting from the expansion of the reconstituting population and the damage to stem cell niches caused by ablative radiation

cell biology↗

bioRxiv2018

Excitable dynamics of Ras triggers self-organized PIP3 signaling for spontaneous cell migration

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. A fundamental question is the molecular mechanism for the spontaneous symmetry breaking. Here we report that GTP bound Ras (Ras-GTP) breaks the symmetry due to excitability even in the absence of extracellular spatial cues and cytoskeletal polarity as well as downstream signaling activities. A stochastic excitation of local and transient Ras activation induced PIP3 accumulation via direct interaction with PI3K, causing tightly coupled traveling waves propagating along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive feedback regulation of Ras-GTP by PIP3. A mathematical model reconstituted a series of the observed symmetry breaking phenomena, illustrating an essential involvement of excitability in the cellular decision-making process.\n\nAuthor contributionsS.F. conducted the experiments; all authors analyzed the data and wrote the manuscript.

cell biology↗

bioRxiv2018

Exosomes are key regulators of non-cell autonomous communication in senescence

Senescence is a cellular phenotype characterized by an irreversible cell cycle arrest and the secretion of inflammatory proteins, denominated senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, until now this role has been mainly attributed to soluble factors. Here, we report that extracellular vesicles also alter the environment by transmitting the senescent phenotype to other cells via exosomes (extracellular vesicles of endocytic origin). A combination of functional assays, Cre-/oxP reporter systems, proteomic analysis and RNAi screens confirm that exosomes form part of the senescent secretome and mediate paracrine senescence via the activation of a non-canonical interferon (IFN) pathway. Altogether, we speculate that exosomes could be drivers of tissue degeneration both locally and systemically during aging and age- related disease.

cell biology↗

bioRxiv2018

Vimentin filaments interact with the mitotic cortex allowing normal cell division

The vimentin network displays remarkable plasticity to support basic cellular functions. Here, we show that in several cell types vimentin filaments redistribute to the cell periphery during mitosis, forming a robust scaffold interwoven with cortical actin and affecting the mitotic cortex properties. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of the tail domain induce increasing impairments of cortical association and mitosis progression. Disruption of actin, but not of microtubules, mimics the impact of tail deletion. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, filament integrity is dispensable for cortical association, which also occurs in vimentin particles. These results unveil novel implications of vimentin dynamics in cell division by means of its interplay with the mitotic cortex.

cell biology↗

bioRxiv2018

Think zinc: Role of zinc poisoning in the intraphagosomal killing of bacteria by the amoeba Dictyostelium

Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit growth of intracellular pathogens. We use the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of non-pathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with \"zincosomes\" of endosomal origin, but also by the action of one or more zinc transporters. We localize the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi apparatus, and study the impact of znt knockouts on zinc homeostasis. Finally, we show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA is killed faster than wild type bacteria.\n\nSummary statementMetal poisoning is one of the bactericidal strategies of macrophages. Here, we describe the dynamics of free Zn and the role of Zn transporters during phagocytosis in Dictyostelium.

cell biology↗