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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,585 records · Page 88Linked to original sources

bioRxiv2018

Host JAK/Stat activity is a target of endoparasitoid wasp virulence strategies

Innate immune responses that allow hosts to survive infection depend on the action of multiple conserved signaling pathways. Pathogens and parasites in turn have evolved virulence factors to target these immune signaling pathways in an attempt to overcome host immunity. Consequently, the interactions between host immune molecules and pathogen virulence factors play an important role in determining the outcome of an infection. The immune responses of Drosophila melanogaster provide a valuable model to understand immune signaling and host-pathogen interactions. Flies are commonly infected by parasitoid wasps and mount a coordinated cellular immune response following infection. This response is characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in the host hemocoel. The conserved JAK-STAT signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Here we show that activity of Stat92E, the D. melanogaster STAT ortholog, is induced in immune tissues following parasitoid infection. Virulent wasp species are able to suppress Stat92E activity during infection, suggesting they target JAK-STAT pathway activation as a virulence strategy. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with a gain-of-function mutation in hopscotch, the D. melanogaster JAK ortholog, indicating that they inhibit the activity of the core signaling components of the JAK-STAT pathway. Our data suggest that parasitoid wasp virulence factors block JAK-STAT signaling to overcome fly immune defenses. Author SummaryFollowing infection, host immune responses are triggered to provide protection against the invading pathogen. The proper function of these responses depends on the activity of multiple immune signaling pathways. These pathways act in a coordinated manner to orchestrate the immune response, and any disruption can render the host susceptible to infection. Because of this sensitivity, many pathogen species have evolved virulence mechanisms that target host signaling pathways and disrupt their function. We are using the interaction between Drosophila melanogaster and Drosophila-infecting parasitoid wasps to study this relationship between host signaling and pathogen virulence. Parasitoids infect fly larvae, and during infection, transfer virulence protein containing venom into the host, providing a mechanism to alter host signaling. This study is focused on one key immune signaling pathway, the highly conserved JAK-STAT pathway. We find that many virulent parasitoid species restrict the activity of the host JAK-STAT pathway, providing a clue as to their virulence strategy. JAK-STAT signaling plays many additional roles in health and disease, and so this system provides a good model to further understand this important pathway.

cell biology↗

bioRxiv2018

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular chemical or physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through mechanosensitive ion channels. This increase in heterochromatin results in increased chromatin-based nuclear rigidity, which suppresses nuclear blebbing in cells with perturbed chromatin or lamins. Furthermore, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics and morphology, including abnormal phenotypes found in human diseases, can be modulated by cell sensing of the extracellular environment and consequent changes to histone modification state and chromatin-based nuclear rigidity, without requiring direct mechanical perturbations to the cell interior.

cell biology↗

bioRxiv2018

Effects of altering histone post-translational modifications on mitotic chromosome structure and mechanics

During cell division chromatin is compacted into mitotic chromosomes to aid faithful segregation of the genome between two daughter cells. Post-translational modifications (PTM) of histones alter compaction of interphase chromatin, but it remains poorly understood how these modifications affect mitotic chromosome stiffness and structure. Using micropipette-based force measurements and epigenetic drugs, we probed the influence of canonical histone PTMs that dictate interphase euchromatin (acetylation) and heterochromatin (methylation) on mitotic chromosome stiffness. By measuring chromosome doubling force (the force required to double chromosome length), we find that histone methylation, but not acetylation, contributes to mitotic structure and stiffness. We discuss our findings in the context of chromatin gel modeling of the large-scale organization of mitotic chromosomes.

cell biology↗

bioRxiv2018

Split-miniSOG for detecting and localizing intracellular protein-protein interactions: application to correlated light and electron microscopy

A protein complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as \"multicolor\" electron microscopy (EM) imaging methods via the photooxidation of 3-3-diaminobenzidine (DAB) and its lanthanide-conjugated derivatives.

cell biology↗

bioRxiv2018

Cell competition corrects noisy Wnt morphogen gradients to achieve robust patterning

Morphogen signaling forms an activity gradient and instructs cell identities in a signaling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signaling and consequent noisy morphogen gradient. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/{beta}-catenin signaling-gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, revealed that unfit cells with abnormal Wnt/{beta}-catenin activity spontaneously appear and produce noise in the Wnt/{beta}-catenin-gradient. Communication between the unfit and neighboring fit cells via cadherin proteins stimulates the apoptosis of the unfit cells by activating Smad signaling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/{beta}-catenin-gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention.

cell biology↗

bioRxiv2018

Dietary fatty acids promote lipid droplet diversity through seipin enrichment in an ER subdomain

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolites-driven targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduced the size of a subset of LDs while over-expression of SEIP-1 had the opposite effect. Ultrastructural analysis revealed SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purified with LDs. Analyses of C. elegans and bacterial genetic mutants indicated a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologous expression of SEIP-1 promoted lipid droplet expansion from ER subdomains in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by enforcing LD diversity.

cell biology↗

bioRxiv2018

A theory of centriole duplication based on self-organized spatial pattern formation

In each cell cycle, centrioles are duplicated to produce a single copy of each pre-existing centriole. At the onset of centriole duplication, the master regulator Polo-like kinase 4 (Plk4) undergoes a dynamic change in its spatial pattern on the periphery of the pre-existing centriole, forming a single duplication site. However, the significance and mechanisms of this pattern transition remain largely unknown. Using super-resolution imaging, we found that centriolar Plk4 exhibits periodic discrete patterns resembling pearl necklaces, frequently with single prominent foci. We constructed mathematical models that simulated the pattern formation of Plk4 to gain insight into the discrete ring patterns. The simulations incorporating the self-organization properties of Plk4 successfully generated the experimentally observed patterns. We therefore propose that the self-patterning of Plk4 is crucial for the regulation of centriole duplication. These results, defining the mechanisms of self-organized regulation, provide a fundamental principle for understanding centriole duplication.

cell biology↗

bioRxiv2018

Functional importance of JMY expression by Sertoli cells in mediating mouse spermatogenesis

Sertoli cells are crucial for spermatogenesis in the seminiferous epithelium because their actin cytoskeleton supports vesicle transport, cell junction, protein anchoring and spermiation. Here, we show that junction-mediating and regulatory protein (JMY), an actin regulating protein, also affects endocytic vesicle trafficking and Sertoli cell junction remodeling since disruption of these functions induced male subfertility in Sertoli cell-specific Jmy knockout mice. Specifically, these mice have: a) impaired BTB integrity and spermatid adhesion in the seminiferous tubules; b) high incidence of sperm structural deformity; c) reduced sperm count and poor sperm motility. Moreover, the cytoskeletal integrity in Sertoli cell-specific Jmy knockout mice was compromised along with endocytic vesicular trafficking. These effects impaired junctional protein recycling and reduced Sertoli cell junctions. In addition, JMY interaction with -actinin1 and Sorbs2 was related to JMY activity and in turn actin cytoskeletal organization. In summary, JMY affects control of spermatogenesis through regulating actin filament organization and endocytic vesicle trafficking in Sertoli cells.

cell biology↗

bioRxiv2018

Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and cell migration

Microtubules play a crucial role in mesenchymal migration by controlling cell polarity and the turnover of cell adhesive structures on the extracellular matrix. The polarized functions of microtubules imply that microtubules are locally regulated. Here, we investigated the regulation and role of two major tubulin post-translational modifications, acetylation and detyrosination, which have been associated with stable microtubules. Using primary astrocytes in a wound healing assay, we show that these tubulin modifications are independently regulated during cell polarization and differently affect cell migration. In contrast to microtubule detyrosination, TAT1-mediated microtubule acetylation increases in the vicinity of focal adhesions and promotes cell migration. We further demonstrate that TAT1 increases focal adhesion turnover by promoting Rab6-positive vesicle fusion at focal adhesions. Our results highlight the specificity of microtubule post-translational modifications and bring new insight into the regulatory functions of tubulin acetylation.

cell biology↗

bioRxiv2018

A molecular explanation for PP2A-B56 isoform specificity during mitosis

PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are shared between B56 isoforms which localise differentially to either the centromere or kinetochore. The centromeric B56 isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. The molecular basis for this differential localisation can be mapped to a small C-terminal region in B56 that regulates these key interactions and defines whether PP2A-B56 complexes bind to either the centromere or kinetochore. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis.

cell biology↗

bioRxiv2018

Cell type-specific differences in redox regulation and proliferation after low UVA doses

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30-50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation. This effect was cell type and dose specific. In both cell lines the levels of reactive oxygen species and nitric oxide fluctuated with dynamics which were influenced differently by UVA; in Me45 cells decreased proliferation accompanied the changes in the dynamics of H2O2 while in HCT116 cells those of superoxide. Genes coding for proteins engaged in redox systems were expressed differently in each cell line; transcripts for thioredoxin, peroxiredoxin and glutathione peroxidase showed higher expression in HCT116 cells whereas those for glutathione transferases and copper chaperone were more abundant in Me45 cells. We conclude that these two cell types utilize different pathways for regulating their redox status. Many mechanisms engaged in maintaining cellular redox balance have been described. Here we show that the different cellular responses to a stimulus such as a specific dose of UVA may be consequences of the use of different redox control pathways. Assays of superoxide and hydrogen peroxide level changes after exposure to UVA may clarify mechanisms of cellular redox regulation and help in understanding responses to stressing factors.

cell biology↗

bioRxiv2018

Id4 eliminates the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells

Quiescence is essential for the long-term maintenance of adult stem cells and tissue homeostasis. However, how stem cells maintain quiescence is still poorly understood. Here we show that stem cells in the dentate gyrus of the adult hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activation state. We found that the inhibitor of DNA binding protein Id4 suppresses Ascl1 activity in neural stem cell cultures. Id4 sequesters Ascl1 heterodimerisation partner E47, promoting Ascl1 protein degradation and neural stem cell quiescence. Accordingly, elimination of Id4 from stem cells in the adult hippocampus results in abnormal accumulation of Ascl1 protein and premature stem cell activation. We also found that multiple signalling pathways converge on the regulation of Id4 to reduce the activity of hippocampal stem cells. Id4 therefore maintains quiescence of adult neural stem cells, in sharp contrast with its role of promoting the proliferation of embryonic neural progenitors.

cell biology↗

bioRxiv2018

NATF (Native And Tissue-specific Fluorescence): A strategy for bright, tissue-specific GFP labeling of native proteins.

GFP labeling by genome editing can reveal the authentic location of a native protein but is frequently hampered by weak GFP signals and broad expression across a range cell types in multicellular animals. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy which combines CRISPR/Cas-9 and split-GFP to yield bright, cell-specific protein labeling. We use CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 {beta}-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein, OIG-1, and demarcates a receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the C. elegans nervous system.

cell biology↗

bioRxiv2018

Mechanisms of integrin αVβ5 clustering in flat clathrin lattices

Summary statementThis article highlights several molecular mechanisms that result in the assembly of integrin V{beta}5-containing flat clathrin lattices in human keratinocytes.\n\nAbstractThe family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cell-extracellular matrix adhesion. The vitronectin-binding integrin V{beta}5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here we show that in human keratinocytes V{beta}5 is predominant found in FCLs and that formation of the V{beta}5-containing FCLs requires the presence of vitronectin as ligand, calcium, and the clathrin adaptor proteins ARH, Numb, and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of {beta}5 and the cytoplasmic domains of {beta}1 or {beta}3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors V{beta}5 clustering in FAs. Our findings strongly indicate that clustering of integrin V{beta}5 in FCLs is dictated by the {beta}5 subunit cytoplasmic domain, cellular tension, and recruitment of specific adaptor proteins to the {beta}5 subunit cytoplasmic domains.

cell biology↗

bioRxiv2018

Infection-generated electric field in gut epithelium drives bidirectional migration of macrophages

Many bacterial pathogens hijack macrophages to egress from the port of entry to the lymphatic/blood-stream, causing dissemination of life-threatening infections. However, the underlying mechanisms are not well understood. Here, we report that Salmonella infection generates directional electric fields (EF) in the follicle-associated epithelium of mouse cecum. In vitro application of an EF, mimicking the infection-generated electric field (IGEF), induces directional migration of primary mouse macrophages to the anode, which is reversed to the cathode upon Salmonella infection. This infection-dependent directional switch is independent of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. The switch is accompanied by a reduction of sialic acids on glycosylated surface components during phagocytosis of bacteria, which is absent in macrophages challenged by microspheres. Moreover, enzymatic cleavage of terminally exposed sialic acids reduces macrophage surface negativity and severely impairs directional migration of macrophages in response to EF. Based on these findings, we propose that macrophages are attracted to the site of infection by a combination of chemotaxis and galvanotaxis; after phagocytosis of bacteria, surface electrical properties of the macrophage change, and galvanotaxis directs the cells away from the site of infection.\n\nAbbreviationsCFU, colony-forming unit; Con A, Concanavalin A; EF, electric field; FAE, follicle-associated epithelium; GNL, Galanthus Nivalis lectin; IGEF, infection-generated electric field; Ji, electric current density; MAL-2, Maackia Amurensis lectin II; MLN, mesenteric lymph node; MOI, multiplicity of infection; nMFI, normalized mean fluorescence intensity; RCA-1, Ricinus Communis Agglutinin I; SNA, Sambucus Nigra lectin; S. Typhimurium, Salmonella enterica serotype Typhimurium; SPI-1, Salmonella pathogenicity island 1; PDMS, polydimethylsiloxane; TEP, trans-epithelial potential difference; TLR, Toll-like receptors; WGEF, wound-generated electric field

cell biology↗

bioRxiv2018

Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage

Tissue repair requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting proliferation remains poorly understood. Here we find that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie dependent genes (myc, e2f1, or cycE). Myc is even sufficient to induce the endocycle in the post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the mitotic activator, Stg, while genetically depleting fzr. However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage and mitotic errors ensue when cells are forced to proliferate. In conclusion, we find that wound-induced polyploidization enables tissue repair when cell division is not a viable option.

cell biology↗

bioRxiv2018

Cullin3-RING ubiquitin ligases are intimately linked to the unfolded protein response of the endoplasmic reticulum

CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric a brac, Tramtrack and Broad-Complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, appears to play an essential role in export of large cargo molecules like procollagen from the endoplasmic reticulum (ER). It has been suggested that CRL3KLHL12 mono-ubiquitinates SEC31 and mono-ubiquitinated SEC31 increases the dimension of a COPII coat to accommodate the large cargo molecules. As we examined this model, we found that functional CRL3KLHL12 was indeed critical for the assembly of large COPII structures. Interestingly, we noticed that CRL3KLHL12 influences collagen synthesis in human skin fibroblasts (HSFs). Our results also suggest that there is a CRL3KLHL12-independent collagen secretion route in HSFs. In addition, we found that CRL3KLHL12 strongly influences levels of sensors of the unfolded protein response (UPR). Different cell lines reacted differently to CUL3 depletion with respect to UPR regulation. This cell line-dependency appears to rely on a cell line-specific BTB-domain protein(s). Consistent with this idea, depletion of a muscle-specific BTB-domain protein KLHL41 recapitulated the effects of CUL3 depletion in C2C12 myotubes in UPR regulation. Based on these results we propose that CRL3KLHL12 and CRL3KLHL41 are regulators of the UPR.

cell biology↗

bioRxiv2018

The nuclear RNAi factor, NRDE2, prevents the accumulation of DNA damage during mitosis in stressful growth conditions

Organisms have evolved multiple mechanisms to prevent and repair DNA damage to protect the integrity of the genome, particularly under stressful conditions. Unrepaired DNA damage leads to genomic instability, aneuploidy, and an increased risk for cancer. Before the cell can divide, it must repair damaged DNA and it is thought that this process requires global silencing of most transcription. In C. elegans, NRDE-2, in complex with other nuclear factors and guided by small RNA, directs heterochromatin formation and transcriptional silencing of targeted genes. Additionally, when C. elegans are cultivated at high temperatures, NRDE-2 is required to maintain germ line immortality. However, the role of NRDE-2 in maintaining the physical integrity of the genome is not understood. We show here that loss of NRDE2 in either nematode or human cells induces the accumulation of DNA damage specifically under conditions of stress, such as cultivation at a high temperature in C. elegans or Aurora B Kinase oncogenic overexpression in the MCF10A epithelial breast cell line. In addition, we found that NRDE2 interacts with {beta}-actin in unstressed mammalian cells. This interaction is dramatically reduced upon DNA damage. The oligomerization state of nuclear actin alters its association with targets, which in turn, regulates their function. Monomeric nuclear actin binds to heterochromatin remodeling factors and transcriptional activators while filamentous actin has been implicated in DNA repair processes. Here we show that NRDE2 associates with actin only when DNA is intact and the bulk of nuclear actin is monomeric. Thus, NRDE2 may dissociate from actin when it becomes filamentous as a result of DNA damage. This implies that, NRDE2, in its role as a heterochromatin factor, binds to monomeric actin, protecting the genome from DNA damage in stressful conditions. In this way, heterochromatin factors may associate with the actin dependent DNA repair process to allow appropriate mitotic progression and maintain genomic integrity.

cell biology↗