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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,639 records · Page 91Linked to original sources

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↗

bioRxiv2018

A cellular anatomy of the normal adult human prostate and prostatic urethra

A cellular anatomy of normal human organs is essential for solving the cellular origins of disease. We report the first comprehensive cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single cell RNA sequencing and flow cytometry on ~98,000 cells taken from different anatomical regions. Two previously unrecognized epithelial cell types were identified by KRT13 and SCGB1A1 expression and found to be highly similar to hillock and club cells of the proximal lung. It was demonstrated by immunohistochemistry that prostate club and hillock cells are similarly concentrated in the proximal prostate. We also optimized a new flow cytometry antibody panel to improve cell type-specific purification based on newly established cellular markers. The molecular classification, anatomical distribution, and purification methods for each cell type in the human prostate create a powerful new resource for experimental design in human prostate disease.

cell biology↗

bioRxiv2018

Activation of the endoplasmic reticulum unfolded protein response reverses an inflammation-like response to cytoplasmic DNA

Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammation that can lead to tissue degeneration. Simpler in vivo models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathology. Here we show that in DNase II-deficient Caenorhabditis elegans, persistent foreign cytoplasmic DNA leads to systemic tissue degeneration, and we identify impaired protein homeostasis as an inflammatory pathomechanism. This pathological outcome can be alleviated by improving protein homeostasis, either via ectopic induction of the endoplasmic reticulum unfolded protein response (UPRER) or by treatment with N-acetylglucosamine. Our results establish C. elegans as an ancestral metazoan model for studying outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for conditions involving chronic inflammation.

cell biology↗

bioRxiv2018

The ULK1-FBXW5-SEC23B nexus controls autophagy

In response to nutrient deprivation, the cell needs to mobilize an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and a source for LC3 lipidation, COPII (Coat Protein Complex II) localizes to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and promotes autophagosome biogenesis. However, the molecular mechanisms that, in response to starvation, divert COPII from the secretory pathway to the autophagic pathway are largely unknown. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting its degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ERGIC, promoting autophagic flux. Induction of autophagy and localization of both SEC23B and SEC24B to the ERGIC in response to nutrient deprivation are significantly reduced in SEC23B(S186A) knock-in cells. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

cell biology↗

bioRxiv2018

Dynamic regulation of the Trypanosoma brucei transferrin receptor in response to iron starvation is mediated via the 3UTR

The bloodstream form of the parasite Trypanosoma brucei obtains iron from its mammalian host by receptor-mediated endocytosis of host transferrin through its own unique transferrin receptor (TbTfR). Expression of TbTfR rapidly increases upon iron starvation by post-transcriptional regulation through a currently undefined mechanism that is distinct from the mammalian iron response system. We have created reporter cell lines by fusing the TbTfR 3'UTR or a control Aldolase 3UTR to reporter genes encoding GFP or firefly Luciferase, and inserted the fusions into a bloodstream form cell line at a tagged ribosomal RNA locus. Fusion of the TbTfR 3UTR is sufficient to significantly repress the expression of the reporter proteins under normal growth conditions. Under iron starvation conditions we observed upregulation of the TbTfR 3UTR fusions only, with a magnitude and timing consistent with that reported for upregulation of the TbTfR. We conclude that the dynamic regulation of the T. brucei transferrin receptor in response to iron starvation is mediated via its 3UTR, and that the effect is independent of genomic location.

cell biology↗

bioRxiv2018

Degron tagging to label membrane-wrapped objects and probe membrane topology

Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we used a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes. These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.

cell biology↗

bioRxiv2018

Centrosome-nuclear envelope tethering and microtubule motor-based pulling forces collaborate in centrosome positioning during mitotic entry

Centrosome positioning relative to the nucleus and cell shape is highly regulated across cell types, during cell migration and during spindle formation in cell division. Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators including Protein Phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators including Dynein and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome positioning. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size prior to mitotic entry, in turn affecting nuclear envelope-based Dynein density and motor capacity. Using computational simulations, PP2A-B55/ SUR-6 regulation of nuclear size and nuclear envelope Dynein density were both predicted to be required for proper centrosome positioning. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome positioning, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taken together, we propose a model in which centrosomes migrate and are positioned through the concerted action of nuclear envelope-based Dynein pulling forces and cen-trosome-nuclear envelope tethering.

cell biology↗

bioRxiv2018

Telomeric dysfunction triggers an unstable growth arrest leading to irreparable genomic lesions and entry into cellular senescence

Replicative senescence is the permanent growth arrest caused by gradual telomere attrition occurring at each round of genome replication. Critically shortened telomeres lose their protective shelterin complex and t-loop structure revealing uncapped chromosome ends that are recognized as DNA double-strand breaks causing a p53-dependent DNA damage response (DDR) towards proliferation arrest. Because telomeres are heterogeneous in length within a single cell, the number of short telomeres necessary for senescence onset remains ill defined. Using controlled Tin2-mediated shelterin inactivation, we show that telomere uncapping is not sufficient to trigger senescence. While uncapping generates expected telomeric DNA damage detection, the associated weak DDR allows a rapid bypass of the primary growth arrest and re-entry into the cell cycle despite dysfunctional telomeres. During the ensuing mitosis, fused telomeres lead to additional DNA breaks and to genomic instability including chromosomes bridges or micronuclei, which sustain a secondary entry into stable growth arrest. The loss of p53 prevented both primary and secondary growth arrest, leading to amplified genomic instablility. Our results support a new multistep model for entry into telomere-mediated replicative senescence in normal cells, which is not directly induced by telomere uncapping, but rather by an amplification of DNA lesions caused by telomere fusions that leads to permanent irreparable genome damage.

cell biology↗

bioRxiv2018

Assembly of Yeast Spindle Pole Body and its Components Revealed by Electron Cryo-Tomography

Spindle pole body (SPB) is the microtubule organizing center (MTOC) in yeast. It plays essential roles during many cellular processes, ranging from mitosis to karyogamy. Here, we used electron cryo-tomography (cryo-ET) and image processing to study SPB and its component purified from the budding yeast. The 3D images and models of SPB at various cell cycle stages were reconstructed by cryo-ET and were analyzed. The results reveal SPB as a cylindrical shaped structure composed of multiple layers. The central layers are arranged with a degree of crystalline order. By using subtomogram averaging methods, we studied the purified \"sheet\" from over-expressed Spc42p. Our analysis of the SPBs and its components provides new insights into the assembly of this organelle and its cellular function as an MTOC.

cell biology↗

bioRxiv2018

Electron Cryo-Tomography Provides Insight into Procentriole Architecture and Assembly Mechanism

Centriole is an essential structure with multiple functions in cellular processes. Centriole biogenesis and homeostasis is tightly regulated. Using electron cryo-tomography (cryoET) we present the structure of procentrioles from Chlamydomonas reinhardtii. We identified a set of non-tubulin components attached to the triplet microtubule (MT), many are at the junctions of tubules likely to reinforce the triplet. We describe structure of the A-C linker that bridges neighboring triplets. We find that POC1 is an integral component of the A-C linker. Its conserved WD40 {beta}-propeller domain provides sites for attachment to other A-C linker components. The twist of A-C linker results in an iris diaphragm-like motion of the triplets in the longitudinal direction of procentriole. Finally, we identified two assembly intermediates at the growing ends of procentriole allowing us to propose a model for the procentriole assembly. Our results provide a comprehensive structural framework for understanding the molecular mechanisms underpinning procentriole biogenesis and assembly.

cell biology↗

bioRxiv2018

DNA damage dependent hypomethylation regulates the pro-angiogenic LncRNA MEG9

Changes in gene expression are key for the cells to adapt and response to intrinsic and extrinsic stimulus. It has been shown that genotoxic stress induces global hypomethylation as a result of decreased expression of DNA methyl transferases (DNMT). We hypothesized that DNA damage suppresses long non-coding RNA expression in the vasculature via DNA methylation leading to more robust DNA repair/survival or cellular senescence/death cell fate decisions. We show here that ionizing radiation reduces the expression of DNMTs in the vascular endothelium and this leads to increased expression of the anti-apoptotic lncRNA MEG9. MEG9 is a lncRNA from the DLK1-DIO3 ncRNA cluster. Loss-of-function studies using RNA gapmers indicate that MEG9 protects endothelial cells from DNA damage induced cell death. Consistent with this phenotype, knockdown of MEG9 decreases growth factor dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Mechanistically, we observed that MEG9 knockdown decreased the expression of cell survival genes including survivin and induced the expression of pro-apoptotic genes such as Bad/Bax. Taken together, our findings illustrate how DNA methylation at selective lncRNA loci can regulate their expression and drive endothelial cell fate decisions.

cell biology↗

bioRxiv2018

Microridges are apical projections formed of branched F-actin networks that organize the glycan layer

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, grow laterally long, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that a microridge is formed of a network of F-actin and requires the function of the Arp2/3 complex for its maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

cell biology↗

bioRxiv2018

Asi1 regulates the distribution of proteins at the inner nuclear membrane in Saccharomyces cerevisiae

Inner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape and stability. Mechanisms that drive changes in the INM proteome are poorly understood in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation (ERAD) and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most pronounced effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggests that Asi1 not only removes mis-targeted proteins at the INM, but it also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33. Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the NE through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.

cell biology↗

bioRxiv2018

A role for long-range, through-lattice coupling in microtubule catastrophe

Microtubules are cylindrical polymers of {beta}-tubulin that play critical roles in fundamental processes like chromosome segregation and vesicular transport. Microtubules display dynamic instability, switching stochastically between growing and rapid shrinking as a consequence of GTPase activity in the lattice. The molecular mechanisms behind microtubule catastrophe, the switch from growing to rapid shrinking, remain poorly defined. Indeed, two-state stochastic models that seek to describe microtubule dynamics purely in terms of the biochemical properties of GTP- and GDP-bound {beta}-tubulin incorrectly predict the concentration-dependence of microtubule catastrophe. Recent studies provided evidence for three distinct conformations of {beta}-tubulin in the lattice that likely correspond to GTP, GDP.Pi, and GDP. The incommensurate lattices observed for these different conformations raises the possibility that in a mixed nucleotide state lattice, neighboring tubulin dimers might modulate each others conformations and hence their biochemistry. We explored whether incorporating a GDP.Pi state or the likely effects of conformational accommodation can improve predictions of catastrophe. Adding a GDP.Pi intermediate did not improve the model. In contrast, adding neighbor-dependent modulation of tubulin biochemistry improved predictions of catastrophe. Conformational accommodation should propagate beyond nearest-neighbor contacts, and consequently our modeling demonstrates that long-range, through-lattice effects are important determinants of microtubule catastrophe.

cell biology↗

bioRxiv2018

Cell Mechanics at the Rear Act To Steer the Direction of Cell Migration

Motile cells navigate complex environments by changing their direction of travel, generating left-right asymmetries in their mechanical subsystems to physically turn. Currently little is known about how external directional cues are propagated along the length scale of the whole cell and integrated with its force-generating apparatus to steer migration mechanically. We examine the mechanics of spontaneous cell turning in fish epidermal keratocytes and find that the mechanical asymmetries responsible for turning behavior predominate at the rear of the cell, where there is asymmetric centripetal actin flow. Using experimental perturbations we identify two linked feedback loops connecting myosin II contractility, adhesion strength and actin network flow in turning cells that are sufficient to recreate observed cell shapes and trajectories in a computational model. Surprisingly, asymmetries in actin polymerization at the cell leading edge play only a minor role in the mechanics of cell turning - that is, cells steer from the rear.\n\nHighlightsO_LIFish keratocytes can migrate with persistent angular velocity, straight or in circles.\nC_LIO_LIAsymmetry in protrusion at the leading edge is not sufficient to generate persistent turning.\nC_LIO_LIAsymmetries in myosin II contraction, actin flow and adhesion at the cell rear cause turns.\nC_LIO_LIOur new computational model of migration predicts observed cell trajectories.\nC_LI

cell biology↗

bioRxiv2018

4D imaging and analysis of multicellular tumour spheroid cell migration and invasion

Studying and characterising tumour cell migration is critical for understanding disease progression and for assessing drug efficacy. Whilst tumour cell migration occurs fundamentally in 3 spatial dimensions (3D), for practical reasons, most migration studies to date have performed analysis in 2D. Here we imaged live multicellular tumour spheroids with lightsheet fluorescence microscopy to determine cellular migration and invasion in 3D over time (4D). We focused on glioblastoma, which are aggressive brain tumours, where cell invasion into the surrounding normal brain remains a major clinical challenge. We developed a workflow for analysing complex 3D cell movement, taking into account migration within the spheroid as well as invasion into the surrounding matrix. This provided metrics characterising cell motion, which we used to evaluate the efficacy of chemother-apeutics on invasion. These rich datasets open avenues for further studies on drug efficacy, microenvironment composition, as well as collective cell migration and metastatic potential.

cell biology↗

bioRxiv2018

The COMA complex is required for positioning Ipl1 activity proximal to Cse4 nucleosomes in budding yeast

Kinetochores are macromolecular protein complexes assembled on centromeric chromatin that ensure accurate chromosome segregation by linking DNA to spindle microtubules and integrating safeguard mechanisms. A kinetochore-associated pool of Ipl1Aurora B kinase, a subunit of the chromosomal passenger complex (CPC), was previously implicated in feedback control mechanisms. To study the kinetochore subunit connectivity built on budding yeast point centromeres and its CPC interactions we performed crosslink-guided in vitro reconstitution. The Ame1/Okp1CENP-U/Q heterodimer, forming the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus and thereby establishes a direct link to the outer kinetochore MTW1 complex. The Sli15/Ipl1INCENP/Aurora B core-CPC interacted with COMA through the Ctf19 C-terminus, and artificial tethering of Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19/Mcm21 deletion in a Sli15 centromere-targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and the relevance of its Sli15/Ipl1 interaction for CPC function.

cell biology↗