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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 343 records · Page 19Linked to original sources

bioRxiv2016

Chytrid fungi construct actin-rich pseudopods, implicating actin regulators WASP and SCAR in an ancient mode of cell motility

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode, and identify its phenotypic and molecular markers. Here, we focus on a widely dispersed migration mode characterized by dynamic, actin-filled pseudopods that we call \"-motility.\" Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE--activators of branched actin assembly--make actin-filled pseudopods. While SCAR has been shown to drive pseudopod formation, WASPs role in this process is controversial. We hypothesize that these genes together represent a genetic signature of -motility, because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration, and colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts -motility in disease-causing chytrid fungi, which we show crawl at >30 m/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of -motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

Cell Biology↗

bioRxiv2016

Robust Gap Repair in the Contractile Ring Ensures Timely Completion of Cytokinesis

Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting contractile rings in C. elegans embryos. Laser cutting causes rings to snap open, which is a sign of tension release. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms that is proportional to ring perimeters before cutting, demonstrating that tension along the ring decreases throughout constriction. Severed rings repair their gaps by recruiting new material and subsequently increase constriction rate and complete cytokinesis with the same timing as uncut rings. Rings repair successive cuts and exhibit substantial constriction when gap repair is prevented. Our analysis suggests that cytokinesis is accomplished by contractile modules that assemble and contract autonomously, enabling local repair of the actomyosin network throughout constriction. Consequently, cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure.

Cell Biology↗

bioRxiv2016

Cell-to-cell spread of microsporidia causes C. elegans organs to form syncytia

The growth of pathogens is dictated by their interactions with the host environment. Many obligate intracellular pathogens undergo several cellular decisions as they progress through their life cycles inside of host cells. We studied this process for several species of microsporidia in the genus Nematocida in their co-evolved animal host Caenorhabditis elegans. We found that microsporidia can restructure multicellular host tissues into a single contiguous multinucleate cell. In particular, we found that all three Nematocida species we studied were able to spread across the cells of C. elegans tissues before forming spores, with two species causing syncytial formation in the intestine, and one species causing syncytial formation in the muscle. We also found that the decision to switch from replication to differentiation in N. parisii was altered by the density of infection, suggesting that environmental cues influence the dynamics of the pathogen life cycle. These findings show how microsporidia can maximize the use of host space for growth, and that environmental cues in the host can regulate a developmental switch in the pathogen.

Cell Biology↗

bioRxiv2016

Harnessing Molecular Motors for Nanoscale Pulldown in Live Cells

Protein-protein interactions (PPIs) regulate signal transduction and cellular behavior, yet studying PPIs within live cells remains fundamentally challenging. We have miniaturized the affinity pulldown, a gold-standard PPI interrogation technique, for use within live cells. Our assay hijacks endogenous myosin motors to forcibly traffic, or pulldown, macromolecular complexes within the native cytosolic environment. Macromolecules captured by nanoscale pulldown (NanoSPD) are optically interrogated in situ by tagging individual protein components. Critically, continuous motor trafficking concentrates query complexes into nanoscopic subcellular compartments, providing fluorescence enhancement and allowing nanoscale pulldowns to be visualized and quantified by standard microscopy. Nanoscale pulldown is compatible with nuclear, membrane-associated and cytoplasmic proteins and can investigate functional effects of protein truncations or amino acid substitutions. Moreover, binding hierarchies in larger complexes can be quickly examined within the natural cytosol, making nanoscale pulldown a powerful new optical platform for quantitative high-content screening of known and novel PPIs that act within macromolecular assemblies.

Cell Biology↗

bioRxiv2016

An improved reversibly dimerizing mutant of the FK506-binding protein FKBP

FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.

Cell Biology↗

bioRxiv2016

NCS1 regulates Ca2+-Dependent Focal Exocytosis of Golgi-derived Vesicles to Help Phagocytic uptake in Macrophages

During phagocytic uptake by macrophages, role of Golgi apparatus was previously ruled out. Notably all such reports were limited to Fc{gamma}-receptor mediated phagocytosis. Here we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos except IgG-coated ones. We report recruitment of Mannosidase-II positive Golgi-derived vesicles during uptake of diverse targets including latex beads, E. coli, Salmonella Typhimurium and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of Mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3 and dynamin-2. Brefeldin A treatment inhibited Mannosidase-II recruitment and phagocytic uptake of serum coated or uncoated latex beads and E. coli. However consistent with previous studies, Brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically recruitment of Mannosidase-II vesicles during phagocytic uptake required Ca2+ from both extra and intra-cellular sources apart from PI3Kinase, microtubules and dynamin-2. Extracellular Ca2+ via voltage-gated Ca2+ channels establish a Ca2+-dependent local PIP3 gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1 however recruitment of recycling endosome marker VAMP3 was unaffected. Both VAMP2 and NCS1 depletion individually resulted in the reduced uptake by macrophages. Together the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function.

Cell Biology↗

bioRxiv2016

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5P and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

Cell Biology↗

bioRxiv2016

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the [~]20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9/sgRNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.\n\nSIGNIFICANCE STATEMENTThe function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome.

Cell Biology↗

bioRxiv2016

Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

Chromatin organization has an important role in the regulation of eukaryotic systems. While recent studies have refined the 3-D models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin -- nucleosomes -- are positioned in 3-D in vivo. Here we have used electron cryotomography to study chromatin organization in the budding yeast S. cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm chromatin fibers. Aside from pre-ribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an \"open\" configuration that is conducive to high levels of transcription. In the absence of higher-order chromatin packing, we propose that yeast can regulate its transcription using local nucleosome-nucleosome associations.

Cell Biology↗

bioRxiv2016

PRDM9 forms a multiprotein complex tethering recombination hotspots to the chromosomal axis

In mammals, meiotic recombination occurs at 1-2 kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We now show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and co-immunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9s KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are co-expressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.

Cell Biology↗

bioRxiv2016

Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes.

Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the nephrocyte architecture and elements that maintain the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Racl and Cdc42 activity as well as proper microtubule organization is required for positioning the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.

Cell Biology↗

bioRxiv2016

KIF1A/UNC-104 transports ATG-9 to regulate neurodevelopment and autophagy at synapses

Autophagy is a cellular degradation process essential for neuronal development and survival. Neurons are highly polarized cells in which autophagosome biogenesis is spatially compartmentalized. The mechanisms and physiological importance of this spatial compartmentalization of autophagy in the neuronal development of living animals are not well understood. Here we determine that, in C. elegans neurons, autophagosomes form near synapses and are required for neurodevelopment. We first determined, through unbiased genetic screens and systematic genetic analyses, that autophagy is required cell-autonomously for presynaptic assembly and for axon outgrowth dynamics in specific neurons. We observe autophagosomes in the axon near synapses, and this localization depends on the synaptic vesicle kinesin, KIF1A/UNC-104. KIF1A/UNC-104 coordinates localized autophagosome formation by regulating the transport of the integral membrane autophagy protein, ATG-9. Our findings indicate that autophagy is spatially regulated in neurons through the transport of ATG-9 by KIF1A/UNC-104 to regulate neurodevelopment.

Cell Biology↗

bioRxiv2016

HIV-1 gag-pol mRNA localization regulates the site of virion assembly

HIV-1 full-length, unspliced genomic RNAs (gRNAs) serve both as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as the genetic material packaged by Gag into virions that assemble at the plasma membrane (PM). Whether localized Gag synthesis contributes to assembly at the PM is unknown. Here we show that artificially tethering gRNAs or surrogate gag-pol mRNAs to non-PM membranes or the actin cytoskeleton can markedly affect Gags distribution in the cytoplasm, causing aberrant subcellular sites of assembly and severe reductions to virus particle output. Only gag-pol mRNAs competent for translation were capable of altering Gags distribution within the cell, and the activity mapped to two cis-acting RNA regulatory elements; the 5 packaging signal (Psi) bound by Gag and, unexpectedly, the Rev response element (RRE) that regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, our results suggest a model wherein localized translation of gRNAs at the PM helps to compartmentalize Gag-gRNA interactions, thereby promoting efficient genome encapsidation.\n\nAUTHOR SUMMARYThe spatial distribution of messenger RNAs (mRNAs) within the cytoplasm can be a crucial determinant of gene expression. Here we provide evidence that a devastating viral pathogen, human immunodeficiency virus type 1 (HIV-1), exploits localized translation to favor the formation of infectious, transmissible virions at the surface of infected cells. Artificially tethering viral mRNAs encoding the Gag and Gag-Pol capsid proteins (gag-pol mRNAs) to alternative regions of the cell such as cytoplasmic vesicles or the actin cytoskeletion markedly alters Gag subcellular distribution, perturbs sites of assembly, and reduces virus particle production. These and additional findings suggest a model for HIV-1 assembly wherein localized Gag/Gag-Pol translation coupled to confined interactions between Gag and viral genomes ensures infectious virion production at the right place and the right time. Perturbing HIV-1 mRNA subcellular localization could represent a novel antiviral strategy.

Cell Biology↗

bioRxiv2016

Centrosome centering and decentering by microtubule network rearrangement

The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs, and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, while the number, dynamics and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized either by robust centrosome centering, robust off-centering or \"reactive\" positioning. In those latter conditions weak asymmetric cues can induce a misbalance of pushing and pulling forces resulting in an abrupt transition from a centered to an off-centered position. Altogether these results point at the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization, but rather as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery.

Cell Biology↗

bioRxiv2016

Gene expression changes after heat shock of procyclic-form Trypanosoma brucei suggest that stress has a role in differentiation to mammalian-infective forms.

Trypanosome procyclic forms multiply in the midgut of Tsetse flies, and are routinely cultured at 27{degrees}C. Heat shocks of 37{degrees}C and above result in general inhibition of translation, and severe heat shock (41{degrees}C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition.a At 27{degrees}C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37{degrees}C - 41{degrees}C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27C and after 1 hour at 39{degrees}C, and the mRNA content of 41{degrees}C heat shocks granules. We found that that mRNAs that bind to ZC3H11 remained in polysomes at 39{degrees}C and were protected from sequestration in granules at 41{degrees}C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. Seventy mRNAs moved towards the polysomal fraction after the 39{degrees}C heat shock; surprisingly, many of these are also increased when trypanosomes migrate to the Tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms\n\nAuthor summaryWhen trypanosomes are inside tsetse flies, they have to cope with temperature variations from below 20{degrees}C up to nearly 40{degrees}C, due to diurnal variations and periodic intake of warm blood. The procyclic forms, which usually multiply in the midgut, are routinely cultured at 27{degrees}C in the laboratory. When they are heated to temperatures of 37{degrees}C and above, they shut down protein production, and at 41{degrees}C, mRNAs aggregate into granules. We show here that quite a large number of mRNAs are not included in granules and continue to be used for making proteins. Some of the proteins that continue to be made are needed in order to defend the cells against the effects of heat shock. Interestingly, however, a moderate heat shock stimulates expression of genes needed for the parasites to develop further into forms that can colonise the salivary glands. It thus seems possible that in the field, temperature variations might influence the efficiency with which of trypanosomes in tsetse flies become infective for mammals.

Cell Biology↗

bioRxiv2016

Zika Virus in the Human Placenta and Developing Brain: Cell Tropism and Drug Inhibition

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including placental damage and microcephaly. However, the placentas role in viral transmission and the mechanisms of microcephaly have not been addressed in primary human tissues. Moreover, there is an urgent need for drugs that can prevent developmental defects following infection. Here, we identify the placental and brain cell populations most susceptible to ZIKV infection, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by inhibiting viral proliferation. In the early gestation placenta, the virus readily infected trophoblast subpopulations that are in direct contact with maternal blood and uterine cells, suggesting routes of ZIKV transmission to the embryo and fetus. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate and brain abnormalities caused by third trimester infection. Blocking a putative viral entry receptor, AXL, which is highly enriched in the infected placenta and brain cell types, reduced ZIKV infection of astrocytes in vitro. In a glial cell line, the macrolide antibiotic, azithromycin, inhibited viral proliferation and viral-induced cytopathic effects at clinically relevant concentrations. Our characterization of infection in primary human tissues clarifies the pathogenesis of congenital ZIKV infection and provides critical context for interpreting results from model systems. Further work on azithromycin and related compounds may yield additional therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Cell Biology↗

bioRxiv2016

The ciliary targeting of membrane proteins by a ternary complex comprising transportin1, Rab8 and the ciliary targeting signal

The sensory functions of cilia are dependent on the enrichment of ciliary resident proteins. While it is known that ciliary targeting signals (CTSs) specifically target ciliary proteins to cilia, it is still unclear how CTSs facilitate the entry and retention of ciliary residents at the molecular level. We found that non-ciliary membrane reporters can passively diffuse to cilia via the lateral transport pathway and the translocation of membrane reporters through the ciliary diffusion barrier is facilitated by importin binding motifs/domains. Screening known CTSs of ciliary membrane residents uncovered that fibrocystin, photoreceptor retinol dehydrogenase, rhodopsin and retinitis pigmentosa 2 interact with transportin1 (TNPO1) via previously identified CTSs. We further discovered that a novel ternary complex, comprising TNPO1, Rab8 and CTS, can assemble or disassemble under the guanine nucleotide exchange of Rab8. Our study suggests a novel mechanism in which TNPO1/Rab8/CTS complex mediates selective entry and retention of cargos within cilia.

Cell Biology↗

bioRxiv2016

Disordered clusters of Bak dimers rupture mitochondria during apoptosis

During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered, compact clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore.

Cell Biology↗