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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,621 records · Page 90Linked to original sources

bioRxiv2018

Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex

ABSTRUCTCondensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H) and two HEAT-repeat (CAP-D2 and -G) subunits. Although it has been shown that balancing acts of the two HEAT-repeat subunits enable this complex to support dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT repeat domain of hCAP-G. A physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only a double-stranded DNA, but also a single-stranded DNA, implicating potential, functional divergence of the vertebrate condensin I complex in mitotic chromosome assembly.

cell biology↗

bioRxiv2018

Combined strategy of siRNA and osteoclast actin cytoskeleton automated imaging to identify novel regulators of bone resorption shows a non-mitotic function for anillin.

Osteoclasts are the main cells responsible for the resorption of mineralized extracellular matrices. They are the major targets for anti-resorptive therapies to manage osteoporosis, a major public health problem. Osteoclasts are giant multinucleated cells that can organize their a unique adhesion structure based on a belt of podosomes, which is the keystone of the bone resorption apparatus. We combined differential transcriptomics and siRNA screening approaches to get a broader view of cytoskeletal regulators that participate in the control of osteoclast cytoskeleton and identify novel regulators of bone resorption by osteoclasts. We identified 20 new candidate regulators of osteoclasts cytoskeleton including Fkbp15, Spire1, Tacc2 and RalA, for which we confirmed they are necessary for proper organization of the podosome belt. We also showed that Anillin, well known for its role during cytokinesis, is essential in osteoclasts for correct podosome patterning and efficient bone resorption. In particular, Anillin controls the levels of the GTPase RhoA, a known regulator of osteoclast cytoskeleton and resorption activity. Finally, we set up and validated an automated imaging strategy based on open-source software for automatic and objective measurement of actin cytoskeleton organization in osteoclasts. We provide these pipelines that are useful to automatically assess the effect of collections of siRNAs or chemical compounds on osteoclast cytoskeleton or differentiation.

cell biology↗

bioRxiv2018

Heterotypic inter-GPCR β-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) activate G protein-coupled receptors (GPCRs) to regulate key pathobiological processes. Here we report a novel lipid mediator GPCR cross-talk mechanism that modulates lymphatic endothelial junctional architecture in lymph nodes. LPAR1 was identified as an inducer of S1PR1/ {beta}-arrestin coupling from a genome-wide CRISPR/ Cas9 transcriptional activation screen. LPAR1 activation induced S1PR1 {beta}-arrestin recruitment while suppressing Gi protein signaling. Lymphatic endothelial cells from cortical and medullary sinuses of lymph nodes which express LPAR1 and S1PR1, exhibit porous junctional architecture and constitutive S1PR1 coupling to {beta}-arrestin which was suppressed by the LPAR1 antagonist AM095. In endothelial cells, LPAR1-activation increased trans-endothelial permeability and junctional remodeling from zipper-like structures to puncta of adhesion plaques that terminate at actin-rich stress fibers with abundant intercellular gaps. Cross-talk between LPA and S1P receptors regulates complex junctional architecture of lymphatic sinus endothelial cells, a site of high lymphocyte traffic and lymph flow.

cell biology↗

bioRxiv2018

Ligand Geometry Controls Adhesion formation via Integrin Clustering

Integrin-mediated cell matrix adhesions are key to sensing the geometry and rigidity of the extracellular environment to regulate vital cellular processes. In vivo, the extracellular matrix (ECM) is composed of a fibrous mesh. To understand the geometry that supports adhesion formation on fibrous substrates, we patterned 10 nm gold-palladium single lines or pairs of lines (total width within 100 nm), mimicking thin single ECM fibers or a minimal mesh geometry, respectively and functionalized it with integrin binding ligand Arg-Gly-Asp (RGD). Single lines showed reduced focal adhesion kinase (FAK) recruitment and did not support cell spreading or formation of focal adhesions, despite the presence of a high density of integrin-binding ligands. Using super resolution microscopy, we observed transient integrin clusters on single lines, whereas stable 110 nm integrin clusters formed on pairs of lines similar to those on continuous substrates. This indicated that two-dimensional ligand geometry is required for adhesion formation on rigid substrates. A mechanism to form modular 100nm integrin clusters bridging the minimal fiber mesh would require unliganded integrins. We observed that integrin mutants unable to bind ligand co-clustered with ligand-bound integrins when present in an active extended conformation. Thus, these results indicate that functional integrin clusters are required to form focal adhesions and unliganded integrins can co-cluster to bridge between thin matrix fibers and can form stable integrin adhesions on dense fibrous networks.

cell biology↗

bioRxiv2018

Establishment of murine in vitro blood-brain barrier models using immortalized cell lines: co-cultures of brain endothelial cells, astrocytes, and neurons

Blood-brain barrier (BBB) is a selective barrier formed by the endothelial cells that line cerebral microvessels. It serves as a physical barrier due to the presence of complex tight junctions between adjacent endothelial cells which limits the paracellular movement of most molecules across the BBB. Many in vitro models of the BBB have been established to mimic these in vivo properties with limited success. In this study, we described the properties of a cell-based murine in vitro BBB model in five configurations constructed using immortalized cell lines in a 12-well format Transwell system: murine brain endothelial cells (bEnd.3) grown in a monoculture, or as co-culture in contact with astrocytes, or without contact with astrocytes or neurons, and triple co-culture combining the three cell lines. We found that only contact and triple co-culture model closely mimic the in vivo BBB tightness as evaluated by apparent permeability (Papp) of sucrose and albumin producing the lowest Papp values of 0.56 {+/-} 0.16 x 10-6 cms-1 and 3.30 {+/-} 0.51 x 10-6 cms-1, respectively, obtained in triple co-culture model. Co-culturing of bEnd.3 with astrocytes increased the expression of occludin as shown by western blot analysis, and immunohistochemistry showed an increase in peripheral localization of occludin and claudin-5. In addition, we found conditioned media were able to increase in vitro BBB model tightness through the modulation of tight junction proteins localization. We conclude that the presence of astrocytes and neurons in close proximity to brain endothelial cells is essential to produce a tight in vitro BBB model.

cell biology↗

bioRxiv2018

Defining the RNA Interactome by Total RNA-Associated Protein Purification

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

cell biology↗

bioRxiv2018

FoxM1 drives proximal tubule proliferation during repair from acute kidney injury

The proximal tubule has a remarkable capacity for repair after acute injury but the cellular lineage and molecular mechanisms underlying this repair response have been poorly characterized. Here, we developed a Kim-1-GFPCreERt2 knockin mouse line (Kim-1-GCE), performed genetic lineage analysis after injury and measured the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones co-expressed Kim-1, Vimentin, Sox9 and Ki67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim-1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells account for repair rather than a fixed tubular progenitor. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor FoxM1 was induced early in injury, was required for epithelial proliferation, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair and we reveal a novel EGFR-FoxM1-dependent signaling pathway that drives proliferative repair after injury.

cell biology↗

bioRxiv2018

Down syndrome fibroblasts exhibit diminished autophagic clearance and endosomal dysfunction after serum starvation

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21 (Tri21). This unbalanced karyotype has the ability to produce proteotoxic stress and dysfunction of the proteostasis network (PN), which are mechanistically associated with several comorbidities found in the DS phenotype. Autophagy is the cellular process responsible for bulk protein degradation and its impairment could negatively impact protein quality control. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts. Both euploid (CTL) and DS fibroblasts induced autophagy successfully through serum starvation (SS), as evidenced by increased LC3-II abundance in CTL and DS. However, DS cells displayed evidence of autophagolysosome (AL) accumulation and impaired clearance of autophagosome cargo, e.g. accumulation of p62 and NBR1. Similar observations were also present in DS cells from multiple differentiation stages, implicating impeded autophagic degradation as a possible early pathologic mechanism in DS. Lysosomal pH and cathepsin B proteolytic activity were found to not differ in CTL and DS fibroblasts after SS, indicating that lysosomal dysfunction did not appear to contribute to unsuccessful autophagic clearance. Based on these results, we hypothesized that possible interference of the endosomal system with autophagy results in autophagosome fusion with endosomal vesicles and negatively impacts degradation. Consistent with this hypothesis, we observed increased abundance of the recycling endosome marker, Rab11, after SS in DS fibroblasts. Further, colocalization of autophagosome markers with resident proteins of early endosomes, late endosomes and recycling endosomes (Rab11) further support our hypothesis. In summary, our work is consistent with impairment of autophagic flux and general PN dysfunction as candidate mechanisms for pathology in DS.

cell biology↗

bioRxiv2018

DNA damage in telophase leads to coalescence between segregated sister chromatid loci.

The generation of DNA double strand breaks (DSBs) pose a high risk for the maintenance of the genome. Cells repair DSBs through two major mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is usually preferred when a sister chromatid is available, thus cells have coupled the activity of the cycling dependent kinase (CDK) to the selection of HR (Symington et al. 2014). Paradoxically, there is a window in the cell cycle where CDK is high despite a sister chromatid is not physically available for HR: late anaphase/telophase. We have here studied in budding yeast the response to DSBs generated in telophase by means of the radiomimetic drug phleomycin. We first show that phleomycin treatment activates the DNA damage response and leads to a delay in the telophase-to-G1 transition. Outstandingly, we also found a partial reversion of sister chromatid segregation, which includes approximation of spindle pole bodies (SPBs) and sister centromeres, de novo formation of anaphase bridges, trafficking of DNA back and forth through the cytokinetic plane and events of coalescence between segregated sister telomeres. We importantly show that phleomycin promotes a massive change in the structure and dynamic of mitotic microtubules (MTs), which coincides with dephosphorylation and re-localization of kinesin-5 Cin8. We propose that anaphase is not entirely irreversible and that there could still be a window to repair DSBs using the sister chromatid after segregation.

cell biology↗

bioRxiv2018

Cytoskeletal tension actively sustains the T cell immunological synapse

When migratory T cells encounter antigen presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T cell immunity. While the cellular processes underlying synapse formation have been well-characterized, those that maintain the symmetry, and thereby the stability of the synapse remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott -Aldrich syndrome Protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T cell activation, WASP is degraded, leading to cytoskeletal rearrangement and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

cell biology↗

bioRxiv2018

Inhibiting histone acetyltransferase activity rescues differentiation of emerin-null myogenic progenitors

IntroductionEmery-Dreifuss Muscular Dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding emerin cause EDMD1. Our previous studies suggested emerin activation of Histone Deacetylase 3 (HDAC3) to reduce Histone 4-Lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. MethodsPharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test if increased acetylated H4K5 was responsible for the impaired differentiation seen in emerin deficient myogenic progenitors. ResultsNu9056 and L002 rescued impaired differentiation in emerin deficiency. SRT1720, which inhibits the NAD+-dependent deacetylase Sirtuin 1 (SIRT1), failed to rescue myotube formation. DiscussionWe conclude emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.

cell biology↗

bioRxiv2018

Reconstitution of the equilibrium state of dynamic actin networks

Principles of regulation of actin network dimensions, fundamentally important for cell functions, remain unclear. We studied in vitro and in silico the effect of key parameters, actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became globally treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic equilibrium state of actin network emerges from biochemical and structural feedbacks.

cell biology↗

bioRxiv2018

Inhibition of platelet function by targeting the platelet cytoskeleton via the LIMK-signaling pathway.

Actin is highly abundant in platelets, and platelet function is dependent on actin structures. Actin filaments are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and slingshot phosphatase (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that inhibition of LIMK activity may prevent the changes in platelet shape during their activation and therefore their function by controlling the dynamics of Factin. Therefore, inhibition of LIMK activity may represent an attractive new strategy to control and inhibit platelet function; particularly the formation of stable platelet aggregates and thus stable thrombi.

cell biology↗

bioRxiv2018

The mitochondrial copper chaperone COX11 plays an auxiliary role in the defence against oxidative stress

COX11, a protein anchored in the inner mitochondrial membrane, was originally identified as a copper chaperone delivering Cu+ to the cytochrome c oxidase of the respiratory chain. Here, we present evidence that this protein is also involved in the defence against reactive oxygen species. Quantitative PCR analyses in the model plant Arabidopsis thaliana revealed that the level of AtCOX11 mRNA rises under oxidative stress. The unexpected result that AtCOX11 knock-down lines contained less ROS than the wild-type can possibly be explained by the impaired oxidative phosphorylation, resulting in less respiration-dependent ROS formation. Similarly, we observed that yeast Saccharomyces cerevisiae ScCOX11 null mutants produced less ROS than wild-type cells. However, when exposed to oxidative stress, yeast strains overexpressing ScCOX11 or AtCOX11 showed lower ROS levels compared with the control indicating a ROS-detoxifying effect of the COX11 proteins. The additive effect on ROS sensitivity upon deletion of ScCOX11 in addition to the known ROS scavenger gene SOD1 encoding superoxide dismutase 1 corroborates the oxidative stress-relieving function of ScCOX11. Moreover, yeast strains overexpressing soluble versions of either AtCOX11 or ScCOX11 became more resistant against oxidative stress. The importance of three conserved cysteines for the ROS scavenger function became apparent after their deletion that resulted in the loss of ROS resistance. Further studies of strains producing COX11 proteins with individually mutated cysteines indicate that the formation of disulphide bridges might be the underlying mechanism responsible for the antioxidative activity of COX11 proteins. Both AtCOX11 and ScCOX11 apparently partake in oxidative stress defence by directly or indirectly exploiting the redox capacity of their cysteine residues.

cell biology↗

bioRxiv2018

Phase Separation of YAP Reprograms Cells for Long-term YAP Target Gene Expression

Yes-associated Protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a selective set of enhancers for potent target gene activation, but how YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus in response to macromolecular crowding. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized YAPs DNA binding cofactor TEAD1 along with other YAP-related transcription co-activators, including TAZ, and subsequently induced transcription of YAP-specific proliferation genes. Super-resolution imaging using Assay for Transposase Accessible Chromatin with photoactivated localization microscopy (ATAC-PALM) revealed that YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA Polymerase II (Pol II), the accessible chromatin domains later acquired Pol II, producing newly transcribed RNA. Removal of YAPs intrinsically-disordered transcription activation domain (TAD) prevented YAP condensate formation and diminished downstream YAP signaling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

cell biology↗

bioRxiv2018

Chromosome missegregation causes somatic polyploidization in plants

Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore-spindle attachment in animal cells. However, kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants. Here, we carried out a comprehensive characterization of conserved kinetochore components in the moss Physcomitrella patens and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins. Most surprisingly, knock-down of several kinetochore proteins led to polyploidy, not aneuploidy, through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer. The resultant cells, containing two or more nuclei, proceeded to the next cell cycle and eventually developed into polyploid plants. As lagging chromosomes have been observed in various plant species in the wild, our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants.

cell biology↗

bioRxiv2018

Light-evoked activity and BDNF regulate mitochondrial dynamics and mitochondrial localized translation

Mitochondria coordinate diverse functions within neurites, including signaling events for axonal maintenance, and degeneration. However, less is known about the role of mitochondria in axon development and maturation. Here we find that in maturing retinal ganglion cells (RGCs) in vivo, axonal mitochondria increase in size, number, and total area throughout development. We demonstrate through multiple approaches in vivo that the mechanism underlying these mitochondrial changes are dependent on eye opening and associated neuronal activity, which can be mimicked by brain derived neurotrophic factor (BDNF). We report downstream gene and protein expression changes consistent with mitochondrial biogenesis and energetics pathways, and present evidence that the associated transcripts are localized and translated at mitochondria within axons in an activity-dependent manner. Together these data support a novel model for mitochondrial-localized translation in support of intra-axonal mitochondrial dynamics and axonal maturation.

cell biology↗

bioRxiv2018

Interactions between N-terminal modules in MPS1 enable spindle checkpoint silencing

AO_SCPCAPBSTRACTC_SCPCAPFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

cell biology↗