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Results for “Cell Biology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Post-Translational Modifications Soften Intermediate Filaments

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of cytoskeletal proteins, modulation of the network architecture and interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording force-strain curves using optical traps. Partial phosphorylation softens the filaments. We show that binding of the protein 14-3-3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14-3-3 may serve to preserve the softening and thereby the altered cell mechanics. We explain our observation by the additional charges introduced during phosphorylation.

biophysics↗

bioRxiv2020

Production and analysis of a mammalian septin hetero-octamer complex

The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

biochemistry↗

bioRxiv2020

Asymmetric neurogenic commitment of retinal progenitors is regulated via the Notch endocytic pathway

During brain development, progenitor cells need to balance proliferation and differentiation in order to generate different neurons in the correct numbers and proportions. Currently, the patterns of multipotent progenitors’ division that lead to neurogenic entry and the factors that regulate them are not fully understood. We here use the zebrafish retina to address this gap, exploiting the possibilities for quantitative live-imaging. We show that early neurogenic progenitors arise from asymmetric divisions. Notch regulates this asymmetry as when inhibited, symmetric divisions producing two neurogenic progenitors occur. Surprisingly, Notch does not act through an apicobasal activity gradient as previously suggested but through asymmetric inheritance of Sara-positive endosomes. Further, the resulting neurogenic progenitors show cell biological features different from multipotent progenitors, raising the possibility that an intermediate state of progenitors exists in the retina. Our study thus opens new insights into the regulation of proliferative and differentiative events during central nervous system development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Super-elasticity of plasma- and synthetic membranes by coupling of membrane asymmetry and liquid-liquid phase separation

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the initial (or projected) PM area. Biochemically isolated lipids self-organize into membranes, but the extraordinary deformability of the plasma membrane is lost. Pure lipid bilayers are prone to rupture at small (<2-4%) area strains and this limits progress for synthetic reconstitution of cellular features such as migration, phagocytosis and division. Here, we show that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity are obtained. We found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These are recruited by mechanical tension applied to the outer vesicle membrane showing an apparent elastic response. This "super-elastic" response emerges from the interplay of lipid liquid-liquid phase separation and membrane asymmetry. This finding allows for bottom-up engineering of synthetic vesicles that appear over one magnitude softer and with three fold larger deformability than conventional lipid vesicles.

biophysics↗

bioRxiv2020

CETSA MS profiling for a comparative assessment of FDA approved antivirals repurposed for COVID-19 therapy identifies Trip13 as a Remdesivir off-target

The reuse of pre-existing small molecules for a novel emerging disease threat is a rapid measure to discover unknown applications for previously validated therapies. A pertinent and recent example where such strategy could be employed is in the fight against COVID-19. Therapies designed or discovered to target viral proteins also have off-target effects on the host proteome when employed in a complex physiological environment. This study aims to assess these host cell targets for a panel of FDA approved antiviral compounds including Remdesivir, using the cellular thermal shift assay (CETSA(R)) coupled to mass spectrometry (CETSA MS) in non-infected cells. CETSA MS is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners as well as in proteins that in turn interact with the direct targets. Such engagement of host targets by antiviral drugs may contribute to the clinical effect against the virus but can also constitute a liability. We present here a comparative study of CETSA molecular target engagement fingerprints of antiviral drugs to better understand the link between off-targets and efficacy.

pharmacology and toxicology↗

bioRxiv2020

Single-cell lineage and transcriptome reconstruction of metastatic cancer reveals selection of aggressive hybrid EMT states

Metastatic cancer remains largely incurable due to an incomplete understanding of how cancer cells disseminate throughout the body. However, tools for probing metastatic dissemination and associated molecular changes at high resolution are lacking. Here we present multiplexed, activatable, clonal, and subclonal GESTALT (macsGESTALT), an inducible lineage recorder with concurrent single cell readout of transcriptional and phylogenetic information. By integrating multiple copies of combined static barcodes and evolving CRISPR/Cas9 barcodes, macsGESTALT enables clonal tracing and subclonal phylogenetic reconstruction, respectively. High barcode editing and recovery rates produce deep lineage reconstructions, densely annotated with transcriptomic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we reconstruct dissemination of tens-of-thousands of single cancer cells representing 95 clones and over 6,000 unique subclones across multiple distant sites, e.g. liver and lung metastases. Transcriptionally, cells exist along a continuum of epithelial-to-mesenchymal transition (EMT) in vivo with graded changes in associated signaling, metabolic, and regulatory processes. Lineage analysis reveals that from a majority of non-metastatic, highly epithelial clones, a single dominant clone that has progressed along EMT drives the majority of metastasis. Within this dominant clone a parallel process occurs, where a small number of aggressive subclones drive clonal outgrowth. By precisely mapping subclones along the EMT continuum, we find that size and dissemination gradually increase, peaking at late-hybrid EMT states but precipitously falling once subclones are highly mesenchymal. Late-hybrid EMT states are selected from a predominately epithelial ancestral pool, enabling rapid metastasis but also forcing extensive and continuous population bottlenecking. Notably, late-hybrid gene signatures are associated with decreased survival in human pancreatic cancer, while epithelial, early-hybrid, and highly mesenchymal states are not. Our findings illuminate features of metastasis and EMT with the potential for therapeutic exploitation. Ultimately, macsGESTALT provides a powerful, accessible tool for probing cancer and stem cell biology in vivo.

cancer biology↗

bioRxiv2020

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. Additionally, Th2 polarization digresses in the absence of CCT-controlled protein folding resulting paradoxically in continued IFN-{gamma} expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

immunology↗

bioRxiv2020

3D Deconvolution Processing for STEM Cryo-Tomography

The complex environment of biological cells and tissues has motivated development of three dimensional imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryo-scanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles, and then in representative cryo-tomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin. Significance statementElectron tomography is used to reveal the structure of cells in three dimensions. The combination with cryogenic fixation provides a snapshot in time of the living state. However, cryo-tomography normally requires very thin specimens due to image formation by conventional phase contrast transmission electron microscopy (TEM). The thickness constraint can be relaxed considerably by scanning TEM (STEM), yet three-dimensional (3D) reconstruction is still subject to artifacts inherent in the collection of data by tilted projections. We show here that deconvolution algorithms developed for fluorescence microscopy can suppress these artifacts, resulting in significant contrast enhancement. The method is demonstrated by cellular tomography of complex membrane structures, and by segmentation of chromatin into distinct, contiguous domains of heterochromatin and euchromatin at high and low density, respectively.

biophysics↗

bioRxiv2020

A Molecular Taxonomy of Tumors Independent of Tissue-of-Origin

Cancer is a complex disease involving disrupted cellular metabolism, basic biochemical processes, and the microenvironment. However, despite some generally agreed upon unifying principles (Hanahan and Weinberg 2000, 2011), molecular signatures remain largely indistinguishable from tissue-of-origin, presenting a major barrier for precision health and individualized medicine. To address this challenge, we reduce mutation data to disruptions in a select set of pathways relevant to basic cell biology, from DNA replication to cellular communication. Using dimensionality reduction techniques, we assign tumor samples into ten clusters distinct from tissue-of-origin and largely free of bias from mutational burden or clinical stage. We show that the clusters vary in prognosis by modeling relative risk of death by cancer type and cluster. We identify cluster-specific mutations in different tissues, demonstrating that tissue-specific signatures contribute to common cellular phenotypes. Moreover, germline risk genes involved in replication fidelity and genome stability are equally distributed among clusters, contrary to the expectation that such genes are avatars of molecular subtype. We investigate metastatic and non-metastatic pathways, and show that most differences are cluster-specific. Some metastatic pathways from one cluster are cluster-specific pathways from non-metastatic tumors of another cluster, suggesting phenotypic convergence. Taken as a whole, our observations suggest that common driver genes combine with tissue-specific disruptions in tumor-promoting pathways to produce a limited number of distinct molecular phenotypes. Thus, we present a coherent view of global tumor biology, and explain how common cellular dysfunction might arise from tissue-specific mutations.

cancer biology↗

bioRxiv2020

MiREDiBase: a manually curated database of editing events in microRNAs

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase.

bioinformatics↗

bioRxiv2020

Pervasive duplication of tumor suppressors in Afrotherians during the evolution of large bodies and reduced cancer risk

The risk of developing cancer is correlated with body size and lifespan within species. Between species, however, there is no correlation between cancer and either body size or lifespan, indicating that large, long-lived species have evolved enhanced cancer protection mechanisms. Elephants and their relatives (Proboscideans) are a particularly interesting lineage for the exploration of mechanisms underlying the evolution of augmented cancer resistance because they evolved large bodies recently within a clade of smaller bodied species (Afrotherians). Here, we explore the contribution of gene duplication to body size and cancer risk in Afrotherians. Unexpectedly, we found that tumor suppresxssor duplication was pervasive in Afrotherian genomes, rather than restricted to Proboscideans. Proboscideans, however, have duplicates in unique pathways that may underlie some aspects of their remarkable anti-cancer cell biology. These data suggest that duplication of tumor suppressor genes facilitated the evolution of increased body size by compensating for decreasing intrinsic cancer risk.

evolutionary biology↗

bioRxiv2020

Understanding the compositional changes in LB Lennox medium during growth of Escherichia coli DH5α in a 1L bioreactor

Compositional changes in growth medium represents dynamic interplay between cell growth, biomass formation, and energy maintenance, with concomitant decrease in nutrients and increase in secreted metabolites and metabolic byproducts. Such information is important for quantifying microbial physiological response both at the population and cellular level, with respect to understanding subtle differences in microbial growth response, as well as supporting model building efforts in metabolic engineering. With the desire to understand molecular weight changes in components of LB Lennox medium as well as broth fractions present during cultivation of Escherichia coli DH5 (ATCC 53868) at 37 {degrees}C, 400 rpm stirring and 1 VVM aeration in a 1 L bioreactor, this study used a combination of gel filtration chromatography (GFC) and reversed phase high performance liquid chromatography (RP-HPLC) for determining changes to molecular weight of different fractions of the growth medium. Experiment results revealed the difficulty of fractionating the culture broth with RP-HPLC, where no distinct peaks of narrow retention time width were obtained. More importantly, the column used for GFC was unable to differentiate small molecular weight changes on the order of a few tens to few hundred Da through a refractive index detector. Together, GFC and RP-HPLC highlighted the difficulty of fractionating LB Lennox culture broth into different distinct fractions. Finally, the study validated the use of 194 nm as detection wavelength for visualizing the chromatogram of LB Lennox medium eluted from a C-18 reversed phase column during liquid chromatography. Collectively, GFC and RP-HPLC could not fractionate LB Lennox broth of E. coli DH5 into distinct fractions for further analysis by identification techniques such as mass spectrometry. Given the inherent complexity of complex medium such as LB Lennox, clean separation of the medium into every component with high purity may be impossible. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/311373v1_ufig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@2d0861org.highwire.dtl.DTLVardef@814ebcorg.highwire.dtl.DTLVardef@158328borg.highwire.dtl.DTLVardef@15e6844_HPS_FORMAT_FIGEXP M_FIG C_FIG Short descriptionBroad elution profile and lack of distinct peaks in chromatogram of LB Lennox broth at 6 hours post inoculation with Escherichia coli DH5 (ATCC 53868), after attempted fractionation by C-18 reversed phase high performance liquid chromatography (RP-HPLC), revealed that separation could not be achieved with a complex mixture such as a microbiology broth. Specifically, while the objective of understanding compositional changes to culture medium during growth presents tremendous opportunities for determining the dynamic conversion of nutrients into metabolites and byproducts, difficulty of profiling all chemical constituents in a complex broth mixture meant that current model building efforts for metabolic engineering remains primitive with respect to the consortia of metabolic reactions occurring in situ at the cellular level. Subject areasbiotechnology, biochemistry, cell biology, microbiology, analytical chemistry

biochemistry↗

bioRxiv2020

ICP-MS analysis of metal and metalloid concentrations of common microbiological growth media reveals presence of heavy metals

Trace elements such as cobalt and manganese play important roles as cofactors of enzymes, and thus, they collectively impact on biochemistry and cellular metabolism. Hence, it is of importance to gain an understanding of the trace metal and metalloid concentrations of growth medium in order to fully account for the growth performance of the bacterium. But, this aspect of microbial cell cultivation is usually neglected. Advent of instrumented techniques for metals and metalloid analysis such as inductively coupled plasma mass spectrometry (ICP-MS) has significantly facilitated trace element analysis at the parts per billion (ppb) to parts per million (ppm) level in aqueous matrixes free of organic interference. In this work, ICP-MS was utilized as the principal tool for profiling the range of metals and metalloids in different microbiological growth medium ranging from minimal salts medium to complex chemically undefined medium. Growth media examined include: LB Lennox, LB Lennox + 2 g/L glucose, LB Lennox (buffered, 89 mM phosphate), LB Lennox (buffered, 89 mM phosphate) + 6 g/L glucose, formulated medium + 6 g/L glucose, Tryptic Soy Broth, M9 medium, and M9 + 1 g/L yeast extract medium. Results reveal detection of elevated concentrations of chromium, copper and cadmium in different versions of LB Lennox and Tryptic Soy Broth. Concentrations of the above heavy metals in the tens of ppm range meant that the growth media could only support the growth of environmental bacteria with some resistance to heavy metal toxicity. On the other hand, no heavy metals were detected in M9 minimal salts medium or a modified version with supplementation of 1 g/L yeast extract. This indicated that minimal salts media may have less of a problem with heavy metal contamination compared to chemically undefined microbiological growth media. Collectively, the results highlight the essentiality of conducting a comprehensive profiling experiment for detecting different metals and metalloids at trace levels in microbiological growth medium. Such data would in addition to offering a deeper understanding of some peculiar growth behaviour from some microorganisms, may also help identify contamination issue during manufacture that preclude use of the media in cultivating many laboratory domesticated microorganisms. Subject areasbiochemistry, biotechnology, cell biology, microbiology, biochemical engineering,

microbiology↗

bioRxiv2020

MiSiC, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities

Studies of microbial communities by live imaging require new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based segmentation method that automatically segments a wide range of spatially structured bacterial communities with very little parameter adjustment, independent of the imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.

microbiology↗

bioRxiv2020

Multiscale and multimodal reconstruction of cortical structure and function

We present a semi-automated reconstruction of L2/3 mouse primary visual cortex from 3 million cubic microns of electron microscopic images, including pyramidal and inhibitory neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are being made publicly available, along with tools for programmatic and 3D interactive access. The density of synaptic inputs onto inhibitory neurons varies across cell classes and compartments. We uncover a compartment-specific correlation between mitochondrial coverage and synapse density. Frequencies of connectivity motifs in the graph of pyramidal cells are predicted quite accurately from node degrees using the configuration model of random graphs. Cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. These example findings illustrate the resources utility for relating structure and function of cortical circuits as well as for neuronal cell biology.

neuroscience↗

bioRxiv2020

A novel family of secreted proteins linked to plant gall development

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls, and that this results in differential expression of a small number of plant genes. Dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. Bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. Bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants. One Sentence SummaryAphid bicycle genes, which encode diverse secreted proteins, contribute to plant gall development.

genetics↗

bioRxiv2020

The meadow jumping mouse genome and transcriptome suggest mechanisms of hibernation

Hibernating mammals exhibit medically relevant phenotypes, but the genetic basis of hibernation remains poorly understood. Using the meadow jumping mouse (Zapus hudsonius), we investigated the genetic underpinnings of hibernation by uniting experimental and comparative genomic approaches. We assembled a Z. hudsonius genome and identified widespread expression changes during hibernation in genes important for circadian rhythm, membrane fluidity, and cell cycle arrest. Tissue-specific gene expression changes during torpor encompassed Wnt signaling in the brain and structural and transport functions in the kidney brush border. Using genomes from the closely related Zapus oregonus (previously classified as Z. princeps) and leveraging a panel of hibernating and non-hibernating rodents, we found selective pressure on genes involved in feeding behavior, metabolism, and cell biological processes potentially important for function at low body temperature. Leptin stands out with elevated conservation in hibernating rodents, implying a role for this metabolic hormone in triggering fattening and hibernation. These findings illustrate that mammalian hibernation requires adaptation at all levels of organismal form and function and lay the groundwork for future study of hibernation phenotypes.

genomics↗

bioRxiv2020

Signalling Network of Breast Cancer Cells in Response to Progesterone

Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. In this study we use antibody microarrays and phosphoproteomics to provide a dynamic global signalling map that reveals new key regulated proteins and links between previously known and novel pathways. Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, EMT, cell adhesion, as well as transcription factors previously not associated with breast cancer proliferation. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management. (197 w)

molecular biology↗