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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 451 records · Page 25Linked to original sources

bioRxiv2017

Activation of NF-κB by a novel, CDK4-regulated, nucleolar stress response pathway

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-{kappa}B transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-{kappa}B also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-{kappa}B pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-{kappa}B signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-{kappa}B induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-{kappa}B pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-{kappa}B pathway. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-{kappa}B in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-{kappa}B nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

cell biology↗

bioRxiv2017

Senescence associated protein degradation

Senescent cells accumulate with age and contribute to pathologies associated to old age. The senescent program can be induced by pro-cancer stimuli or is developmentally controlled. In cells forced to senesce by expression of oncogenes or short telomeres, aberrant activation of the ERK/MAP kinase signaling pathway leads to selective protein degradation by the ubiquitin proteasome system. The proteins affected by this process control key cellular processes known to be defective in senescent cells. We discuss the evidence supporting a general role for senescence associated protein degradation for organismal aging.

cell biology↗

bioRxiv2017

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterizing transcriptional changes in cord blood derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show, that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the two stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the two phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process, away from a simple binary switch between two options as it is usually envisioned.

cell biology↗

bioRxiv2017

Glycolytic reliance promotes anabolism in photoreceptors

Sensory neurons capture information from the environment and convert it into signals that can greatly impact the survival of an organism. These systems are thus under heavy selective pressure, including for the most efficient use of energy to support their sensitivity and efficiency1. In this regard, the vertebrate photoreceptor cells face a dual challenge. They not only need to preserve their membrane excitability via ion pumps by ATP hydrolysis2 but also maintain a highly membrane rich organelle, the outer segment, which is the primary site of phototransduction, creating a considerable biosynthetic demand. How photoreceptors manage carbon allocation to balance their catabolic and anabolic demands is poorly understood. One metabolic feature of the retina is its ability to convert the majority of its glucose into lactate3,4 even in the presence of oxygen. This phenomenon, aerobic glycolysis, is found in cancer and proliferating cells, and is thought to promote biomass buildup to sustain proliferation5,6. The purpose of aerobic glycolysis in the retina, its relevance to photoreceptor physiology, and its regulation, are not understood. Here, we show that rod photoreceptors rely on glycolysis for their outer segment (OS) biogenesis. Genetic perturbations targeting allostery or key regulatory nodes in the glycolytic pathway impacted the OS size. Fibroblast growth factor (FGF) signaling was found to regulate glycolysis, with antagonism of this pathway resulting in anabolic deficits. These data demonstrate the cell autonomous role of the glycolytic pathway in OS maintenance and provide evidence that aerobic glycolysis is part of a metabolic program that supports the biosynthetic needs of a normal neuronal cell type.

cell biology↗

bioRxiv2017

Human microcephaly ASPM protein is a spindle pole-focusing factor

Nonsense mutations in the ASPM gene have been most frequently identified among familial microcephaly patients. Depletion of the Drosophila orthologue causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, using CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein CDK5RAP2 (also called CEP215) in human tissue culture cells. Deletion of the ASPM gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in ASPM KO cells, spindle poles were unfocused during prometaphase and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET or activate the{gamma} -tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.

cell biology↗

bioRxiv2017

A Molecular Signature for Anastasis, Recovery from the Brink of Apoptotic Cell Death

During apoptosis, executioner caspase activity has been considered a point of no return. However, recent studies show that cells can survive caspase activation following transient apoptotic stimuli, a process named anastasis. To identify a molecular signature, we performed whole transcriptome RNA sequencing of untreated, apoptotic, and recovering HeLa cells. We found that anastasis is an active, two-stage program. During the early stage, cells transition from growth-arrested to growing. In the late stage, cells change from proliferating to migratory. Strikingly, some early recovery mRNAs were elevated first during apoptosis, implying that dying cells poise to recover, even while still under apoptotic stress. Furthermore, TGF{beta}-induced Snail expression is required for anastasis, and recovering cells exhibit prolonged elevation of pro-angiogenic factors. This study demonstrates similarities in the anastasis genes, pathways, and cell behaviors to those activated in wound healing. This study identifies a repertoire of potential targets for therapeutic manipulation of this process.

cell biology↗

bioRxiv2017

Photorelease of Diacylglycerol Increases the Amplitude and Duration of Protein Kinase C-BetaII Relocation in cyto

Diacylglycerol (DAG) is a lipid second messenger produced by receptor stimulated phospholipase C and is capable of activating several PKC isoforms. Classical PKC isoforms require simultaneous presence of calcium and DAG for activation and relocation to membranes. The aim of this study was to synthesize a photolabile precursor of DAG and examine the effect of an immediate increase of the signaling lipid on PKC relocation. Caged DAG was synthesized using a photoreleasable 7-diethyl-aminocoumarin (DEACM) group. Photolysis was monitored in vitro by an increase in coumarin fluorescence from which an uncaging quantum yield of 1.1% was determined. This quantum yield proved ideal for efficient uncaging at high UV power while allowing localization of the fluorescent compound at low UV power. Taking advantage of the coumarin fluorescence, it was demonstrated that DEACM-DiC8 accumulated inside cells. Effects of DAG photorelease on periodic relocations of PKCbetaII, induced by histamine, UTP or EGF, were studied. Photorelease of DAG in cyto immediately increased the amplitude and duration of relocation events, regardless of the agonist used. Together, the results demonstrate the usefulness of caged DAG for dissecting PKC signaling and suggest that DAG levels are limiting during signaling.

cell biology↗

bioRxiv2017

Mechanical activation of epithelial Na+ channel relies on an interdependent activity of the extracellular matrix and extracellular N-glycans of αENaC

Mechanotransduction describes how cells perceive their mechanical environment and mechanosensitive ion channels are important for this process. ENaC (epithelial Na+ channel)/DEG (degenerin) proteins form mechanosensitive ion channels and it is hypothesized their interaction with the extracellular matrix (ECM) via tethers is required for mechanotransduction. Channels formed by vertebrate , {beta} and {gamma} ENaC proteins are activated by shear force (SF) and mediate electrolyte/fluid-homeostasis and blood pressure regulation. Here, we report an interdependent activity of ENaC and the ECM that mediates SF effects in murine arteries and heterologously expressed channels. Furthermore, replacement of conserved extracellular N-glycosylated asparagines of ENaC decreased the SF response indicating that the attached N-glycans provide a connection to the ECM. Insertion of N-glycosylation sites into a channel subunit, innately lacking these motifs, increased its SF response. These experiments confirm an interdependent channel/ECM activity of mechanosensitive ENaC channel and highlight the role of channel N-glycans as new constituents for the translation of mechanical force into cellular signals.

cell biology↗

bioRxiv2017

Initiation of DNA replication requires actin dynamics and formin activity

Nuclear actin influences transcription in a manner dependent on its dynamics of polymerisation and nucleocytoplasmic translocation. Using human somatic cells and transcriptionally-silent Xenopus egg extracts, we show that actin dynamics is also required for DNA replication. We identify many actin regulators in replicating nuclei from Xenopus egg extracts, and show that in human cells, nuclear actin filaments form in early G1 and disassemble prior to S-phase. In either system, treatments that stabilise nuclear actin filaments abrogate nuclear transport and initiation of DNA replication. Mechanistically, actin directly binds RanGTP-importin complexes and disruption of its dynamics hinders cargo release. This prevents both nuclear pore complex (NPC) formation and active nuclear transport, which we show is required throughout DNA replication. Nuclear formin activity is required for two further steps: loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin and initiation of DNA replication. Thus, actin dynamics and formins are involved in several nuclear processes essential for cell proliferation.

cell biology↗

bioRxiv2017

A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion

In non-motile fungi, sexual reproduction relies on stron morphogenetic changes in response to pheromone signaling. We report here on asystematic screen for morphological abnormalities o the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions which, upon visual screening, revealed a plethora of phenotype affecting all stages of the mating process, including cell polarizati cell fusion and sporulation. Cell fusion relies onthe formation of the fusion focus, an aster-like F-actin structure that is marked by stron local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondaryscreen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical forthe coalescence of the fusion focus Indeed, rng8{triangleup} and rng9{triangleup} mutant cells exhibitmultiple stable dots a the cell-cell contact site, instead of the single cusfo observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, depende on Myo51 and tropomyosin Cdc8A. tropomyosin mutant allele, whic compromises Rng8/9 localization but not actin binding, similarly lea to multiple stable dots instead of a single focus.By contrast, myo51 deletion does not strongly affect fusion focus coalescenceWe. propose that focusing of the actinfilaments in the fusionaster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments.

cell biology↗

bioRxiv2017

Mapping load-bearing in the mammalian spindle reveals local kinetochore-fiber anchorage that provides mechanical isolation and redundancy

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore-fibers (k-fibers) to bear this load. We know that the mammalian spindle body can bear the load of chromosome movement far from poles, but do not know where and how - physically and molecularly - this load is distributed across the spindle. In part, this is because perturbing and reading out spindle mechanics in live cells is difficult. Yet, answering this question is key to understanding how the spindle generates and responds to force, and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time, and dissect local anchorage mechanics and mechanism. To do so, we laser ablate single k-fibers at different spindle locations, and in different molecular backgrounds, and quantify at high time resolution the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 m from kinetochores, scaling with k-fiber length, and laterally within ~2 m of k-fiber sides, without neighboring k-fibers sharing load. A phenomenological model constrains the mechanistic underpinnings of these data: it suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement, but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, while Eg5 and PRC1 are not, suggesting specialization in mechanical function and a novel function for NuMA throughout the spindle body. Together, the data and model suggest that widespread NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well-suited to support robust chromosome segregation.

cell biology↗

bioRxiv2017

Multiple kinesin-14 family members drive microtubule minus-end-directed transport in plant cells

Minus-end-directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, plants have lost the genes encoding dynein during evolution; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus-end-directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class-VI kinesin-14, KCBP, is required for minus-end-directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids (PLs) and unidirectionally transported PL liposomes along MTs in vitro. Thus, minus-end-directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus-end-directed motility, and we identified the conserved class-I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus-end-directed transporters in land plants.

cell biology↗

bioRxiv2017

Spindle assembly checkpoint satisfaction occurs through end-on but not lateral kinetochore-microtubule interactions under tension

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. What signals the SAC monitors remains unclear. We do not know the contributions of different microtubule attachment features, or tension from biorientation, to SAC satisfaction in normal mitosis - or how these possible cues change during attachment. Here, we quantify concurrent Mad1 intensity, reporting on SAC silencing, and real-time attachment geometry, occupancy, and tension at individual mammalian kinetochores. We show that Mad1 loss from the kinetochore occurs in switch-like events with robust kinetics, and that metaphase-like tension across sister kinetochores is established just before Mad1 loss events at the first sister. We demonstrate that CenpE-mediated lateral attachment of the second sister can persistently generate this metaphase-like tension prior to biorientation, likely stabilizing sister end-on attachment, yet cannot induce Mad1 loss from that kinetochore. Instead, Mad1 loss begins after several end-on microtubules attach. Thus, end-on attachment provides geometry-specific molecular cues, or force on specific kinetochore linkages, that other attachment geometries cannot provide.\n\nSummaryThe spindle assembly checkpoint (SAC) delays anaphase until kinetochores are properly attached to the spindle. The authors demonstrate that the SAC monitors geometry-specific molecular cues, or force on specific kinetochore linkages, that \"end-on\" but not \"lateral\" attachments generating persistent tension can provide.

cell biology↗

bioRxiv2017

Chromatin tethering to the nuclear envelope by nuclear actin filaments: a novel role of the actin cytoskeleton in the Xenopus blastula

The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the sub-nuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing, large Xenopus blastula cells.

cell biology↗

bioRxiv2017

A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast

In budding yeast, cell cycle progression and ribosome biogenesis are dependent upon plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here, we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network plays an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together, these discoveries provide new clues to how growth-dependent signals control cell growth and the cell cycle.

cell biology↗

bioRxiv2017

The mammalian kinetochore independently regulates its passive and active force-generating interfaces with microtubules

The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore slides along microtubules [3,4]. We do not know the physical and molecular nature [5-7] of these interfaces, or how they are regulated to support diverse mitotic functions in mammalian cells. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8-11]. Hec1s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12-15], but its role at the passive and active interfaces remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1s role as it moves on polymerizing versus depolymerizing microtubules. We show that Hec1 phosphorylation tunes passive friction along polymerizing microtubules, modulating both the magnitude and timescale of responses to force. In contrast, we find that Hec1 phosphorylation does not affect the kinetochores ability to grip depolymerizing microtubules, or switch to this active force-generating state. Together, the data suggest that different kinetochore interfaces engage with growing and shrinking microtubules, and that passive friction can be regulated without disrupting active force generation. Through this mechanism, the kinetochore can modulate its grip on microtubules as its functional needs change during mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

cell biology↗

bioRxiv2017

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naive pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

cell biology↗

bioRxiv2017

Small molecule-mediated reprogramming of epithelial-mesenchymal transition thereby blocking fibrosis

Fibrotic diseases are major causes of morbidity and mortality, and the epithelial-mesenchymal transition (EMT) plays a central role in the development of tissue/organ fibrosis. We discovered that eupatilin, a member of the chromone scaffold (CS)-containing compounds found ubiquitously in the plant kingdom, completely reversed fibrogenesis in vitro and substantially ameliorated bleomycin-induced lung fibrosis (BILF). Furthermore, eupatilin-induced growth arrest and morphological changes in primary fibroblasts derived from a patient with idiopathic pulmonary fibrosis (IPF). To better understand fibrosis, we established a mouse hepatic stellate cell (HSC) line that was robustly differentiated into myofibroblasts upon treatment with TGF{beta}. HSC-derived fibrogenesis was completely blocked by eupatilin, which caused dramatic morphological changes while inhibiting expression of EMT-related genes. The chemical groups linked to the 2nd carbon (C2), C3, C6, and C7 on the CS of eupatilin were essential for its anti-fibrogenic effects. Unlike eupatilin, pirfenidone failed to block HSC fibrogenesis and did not affect the morphology of HSCs or lung fibroblasts. Although pirfenidone affected local production of TGF{beta}, as reflected by a reduction in the TGF{beta} level in lung lysates of BILF model mice, eupatilin is likely to act via a different therapeutic mechanism. In particular, eupatilin had greater anti-fibrotic capacity and EMT-inhibitory activity and significantly attenuated the phosphorylation of Erk by TGF{beta}. Based on the interactome, Integrin{beta}3 seems to be a major player in integration of TGF{beta} signaling into the eupatilin-mediated anti-fibrosis. Our findings suggest that combinatorial use of eupatilin and pirfenidone may augment the therapeutic efficacy of IPF treatment.

cell biology↗