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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 955 records · Page 53Linked to original sources

bioRxiv2020

In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.

plant biology↗

bioRxiv2020

Design of Biologically Active Binary Protein 2D Materials

Proteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9-12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems. One Sentence SummaryCo-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

synthetic biology↗

bioRxiv2019

SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani

Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp affect their host, are not fully understood. Herein, proteomic alterations of primary bone marrow-derived, BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, with the use of a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (>5500 proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis allows new technical insight into the challenges of quantitative proteomics applied on primary cells, and establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface.

systems biology↗

bioRxiv2020

Analyses of Multi-dimensional Single Cell Trajectories Quantify Transition Paths Between Nonequilibrium Steady States

How a cell changes from one stable phenotype to another one is a fundamental problem in developmental and cell biology. Epithelial-to-mesenchymal transition (EMT) is a phenotypic transition process extensively studied recently but mechanistic details remain elusive. Through time-lapse imaging we recorded single cell trajectories of human A549/Vim-RFP cells undergoing EMT induced by different concentrations of TGF-{beta} in a multi-dimensional cell feature space. The trajectories cluster into two distinct groups, indicating that the transition dynamics proceeds through parallel paths. We then reconstructed the reaction coordinates and corresponding pseudo-potentials from the trajectories. The potentials reveal a plausible mechanism for the emergence of the two paths as the original stable epithelial attractor collides with two saddle points sequentially with increased TGF-{beta} concentration, and relaxes to a new one. Functionally the directional saddle-node bifurcation ensures a CPT proceeds towards a specific cell type, as a mechanistic realization of the canalization idea proposed by Waddington.

biophysics↗

bioRxiv2020

SABRE populates ER domains essential for cell plate maturation and cell expansion influencing cell and tissue patterning

The SABRE protein, originally identified in plants, is found throughout eukaryotes. In plants, SABRE has been implicated in cell expansion, division plane orientation and planar polarity. However, how SABRE mediates these processes remains an open question. Here, we have taken advantage of the fact that the bryophyte Physcomitrium patens has a single copy of SABRE, is an excellent model for cell biology and is readily amenable to precise genetic alterations to investigate SABREs mechanism of action. We discovered that SABRE null mutants were stunted in both polarized growing and diffusely growing tissues, similar to reported phenotypes in seed plants. However, in polarized growing cells, we observed significant delays in cell plate formation and sometimes catastrophic failures in cell division. We generated a functional SABRE fluorescent fusion protein and determined that it forms dynamic puncta on regions of the endoplasmic reticulum (ER) both in the cytoplasm during interphase and at the new cell plate during division. In the absence of SABRE, ER morphology was severely compromised with large aggregates accumulating in the cytoplasm and abnormal buckling along the developing cell plate late in cytokinesis. In fact, SABRE and the ER maximally accumulated on the developing plate specifically during cell plate maturation, coincident with the timing of the onset of failures in cell plate formation in cells lacking SABRE. Further we discovered that callose deposition is delayed in{Delta} sabre cells, and in cells that failed to divide, abnormal callose accumulations formed at the cell plate. Our findings demonstrated that SABRE functions by influencing the ER and callose deposition, revealing a surprising and essential role for the ER in cell plate maturation. Given that SABRE is conserved, understanding how SABRE influences cell and tissue patterning has profound significance across eukaryotes.

plant biology↗

bioRxiv2021

Creating wounds in cell monolayers using micro-jets

Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a contactless method to create wounds with any imaginable 2D pattern in cell monolayers using micro-jets of either media or an immiscible and biocompatible fluorocarbon (i.e., FC40). We also combine this with another method that allows automation and multiplexing using standard Petri dishes. A dish is filled with a thin film of media overlaid with FC40, and the two liquids reshaped into an array of microchambers in minutes. Each chamber in such a grid is isolated from others by fluid walls of FC40. Cells are now added, allowed to grow into a monolayer, and wounds created using the microjets; then, healing is monitored by microscopy. As arrays of chambers can be made using the media and Petri dishes familiar to biologists, and as dishes fit seamlessly into their incubators, microscopes, and workflows, we anticipate this assay will find wide application in biomedicine.

bioengineering↗

bioRxiv2024

ProteinWeaver: A Webtool to Visualize Ontology-Annotated Protein Networks

Molecular interaction networks are a vital tool for studying biological systems. While many tools exist that visualize a protein or a pathway within a network, no tool provides the ability for a researcher to consider a proteins position in a network in the context of a specific biological process or pathway. We developed ProteinWeaver, a web-based tool designed to visualize and analyze non-human protein interaction networks by integrating known biological functions. ProteinWeaver provides users with an intuitive interface to situate a user-specified protein in a user-provided biological context (as a Gene Ontology term) in five model organisms. Protein-Weaver also reports the presence of physical and regulatory network motifs within the queried subnetwork and statistics about the proteins distance to the biological process or pathway within the network. These insights can help researchers generate testable hypotheses about the proteins potential role in the process or pathway under study. Two cell biology case studies demonstrate ProteinWeavers potential to generate hypotheses from the queried subnetworks. ProteinWeaver is available at https://proteinweaver.reedcompbio.org/.

systems biology↗

bioRxiv2023

Consideration of sex as a biological variable in diabetes research across twenty years

Sex differences exist in the risk of developing both type 1 and type 2 diabetes, and in the risk of developing diabetes-associated complications. Sex differences in glucose homeostasis, islet and {beta} cell biology, and peripheral insulin sensitivity have also been reported in multiple animals. To determine the degree to which biological sex has been addressed in published literature related to diabetes and insulin biology, we developed a scoring system to assess the inclusion of biological sex in papers related to these topics. We scored manuscripts published in Diabetes, published by the American Diabetes Association, as this journal focuses on diabetes and diabetes-related research. We scored papers published across three years within a 20-year period (1999, 2009, 2019), a timeframe that spans the introduction of funding agency and journal policies to improve the consideration of biological sex as a variable. Our analysis shows fewer than 15% of papers used sex-based analysis in even one figure across all study years, a trend that was reproduced across journal-defined categories of diabetes research (e.g., islet studies, signal transduction). Single-sex studies accounted for approximately 40% of all manuscripts, of which >87% used male subjects only. While we observed a modest increase in the overall inclusion of sex as a biological variable during our study period, our results highlight significant opportunities to improve consideration of sex as a biological variable in diabetes research. In particular, we show that journal policies represent one way to promote better consideration of biological sex as a variable. In the long term, improved practices will reveal sex-specific mechanisms underlying diabetes risk and complications, generating insights to support the development of sex-informed prevention and treatment strategies.

scientific communication and education↗

bioRxiv2023

A rapid method to quantify vein density in C4 plants using starch staining

C4 photosynthesis has evolved multiple times in the angiosperms and typically involves alterations to the biochemistry, cell biology and development of leaves. One common modification found in C4 plants compared with the ancestral C3 state is an increase in vein density such that the leaf contains a larger proportion of bundle sheath cells. Recent findings indicate that there may be significant intra-specific variation in traits such as vein density in C4 plants but to use such natural variation for trait-mapping, rapid phenotyping would be required. Here we report a high-throughput method to quantify vein density that leverages the bundle sheath specific accumulation of starch found in C4 species. Starch staining allowed high-contrast images to be acquired that permitted image analysis using a MATLAB-based program. The method works for the dicotyledon Gynandropsis gynandra where significant variation in vein density was detected between natural accessions, and the monocotyledon Zea mays where no variation was apparent in the genotypically diverse lines assessed. We anticipate this approach will be useful to map genes controlling vein density in C4 species demonstrating natural variation for this trait. One sentence summaryPreferential accumulation of starch in bundle sheath cells of C4 plants allows high-throughput phenotyping of vein density.

plant biology↗

bioRxiv2022

Learning representations for image-based profiling of perturbations

Measuring the phenotypic effect of treatments on cells through imaging assays is an efficient and powerful way of studying cell biology, and requires computational methods for transforming images into quantitative data that highlight phenotypic outcomes. Here, we present an optimized strategy for learning representations of treatment effects from high-throughput imaging data, which follows a causal framework for interpreting results and guiding performance improvements. We use weakly supervised learning (WSL) for modeling associations between images and treatments, and show that it encodes both confounding factors and phenotypic features in the learned representation. To facilitate their separation, we constructed a large training dataset with Cell Painting images from five different studies to maximize experimental diversity, following insights from our causal analysis. Training a WSL model with this dataset successfully improves downstream performance, and produces a reusable convolutional network for image-based profiling, which we call Cell Painting CNN-1. We conducted a comprehensive evaluation of our strategy on three publicly available Cell Painting datasets, discovering that representations obtained by the Cell Painting CNN-1 can improve performance in downstream analysis for biological matching up to 30% with respect to classical features, while also being more computationally efficient.

bioinformatics↗

bioRxiv2016

Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C

BackgroundHuman natural killer (NK) cell activity is regulated by a family of killer-cell Ig-like receptors (KIR) that bind human leucocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys80) and KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn80). However, this model does not capture allelic diversity in HLA-C or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes.\n\nResultsThe impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2, C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides, or predicted HIV Gag peptides, by HLA-C can promote KIR cross-reactive binding.\n\nConclusionsKIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, which provides an explanation for why KIR2DL3-C1 interactions appear weaker than KIR2DL1-C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR-HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.

immunology↗

bioRxiv2021

Endosomal structure and APP biology are not altered in preclinical cellular models of Down syndrome

Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimers disease - dementia. Alzheimers disease is characterised by the accumulation in the brain of amyloid-{beta} plaques that are a product of amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-{beta} accumulation is within endosomes and changes to endosome biology occur early in disease. Here we determine if primary mouse embryonic fibroblasts isolated from two mouse models of Down syndrome can be used to study endosome and APP cell biology. We report that in these cellular models of Down syndrome endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in these models, despite three copies of App.

neuroscience↗

bioRxiv2024

BRD4 phosphorylation regulates the structure of chromatin nanodomains

The interplay between chromatin structure and phase-separating proteins is an emerging topic in cell biology with implications for understanding disease states. Here, we investigate the functional relationship between bromodomain protein 4 (BRD4) and chromatin architecture. By combining molecular dynamics simulations with live-cell imaging, we demonstrate that BRD4, when mutated at specific N-terminus sites, significantly impacts nucleosome nanodomain (NN) organization and dynamics. Our findings reveal that enhanced chromatin binding activity of BRD4 condenses NNs, while both loss or gain of BRD4 chromatin binding reduced diffusion of single nucleosomes, suggesting a role for BRD4 in the regulation of nanoscale chromatin architecture and the chromatin microenvironment. These observations shed light on the nuanced regulation of chromatin structure by BRD4, offering insights into its role in maintaining the nuclear architecture and transcriptional activity.

biophysics↗

bioRxiv2026

Modular presynaptic assemblages scale to postsynaptic partner number

Behavioral diversification can arise through, and is constrained by, evolutionary and inter-individual differences in neural circuit development. Moreover, alteration of focal neural parameters changes the environment in which cells connect into circuits. In the mushroom body, an associative learning center of arthropods, the number of principal Kenyon cells varies widely across species and among individuals. How such variation is developmentally accommodated by projection neurons, which provide sensory input to Kenyon cells, is not understood. In Drosophila melanogaster, we previously demonstrated that projection neurons scale their presynaptic bouton number to Kenyon cell population size. Here, we identify the developmental mechanisms underlying this input flexibility. Boutons arise from projection neuron axonal collaterals; we find that a PNs collateral number is subtype-specific and serves as the substrate through which bouton number scales to Kenyon cell population size. Independent of projection neuron identity or Kenyon cell number, individual collaterals most often produce just one bouton, suggesting collaterals are modular cell biological bouton units. Developing projection neurons initially overproduce nascent collaterals in the early pupa. The set of nascent collaterals that mature and eventually bear boutons is conditional on Kenyon cell number, thereby executing scaling. Finally, early boutons bear filopodia that frequently contact neighboring PN processes, suggesting that bouton-bouton interactions contribute to shaping these structures.

neuroscience↗

bioRxiv2024

Pathway metabolite ratios reveal distinctive glutamine metabolism in a subset of proliferating cells

Large-scale metabolomic analyses of pan-cancer cell line panels have provided significant insights into the relationships between metabolism and cancer cell biology. Here, we took a pathway-centric approach by transforming targeted metabolomic data into ratios to study associations between reactant and product metabolites in a panel of cancer and non-cancer cell lines. We identified five clusters of cells from various tissue origins. Of these, cells in Cluster 4 had high ratios of TCA cycle metabolites relative to pyruvate, produced more lactate yet consumed less glucose and glutamine, and greater OXPHOS activity compared to Cluster 3 cells with low TCA cycle metabolite ratios. This was due to more glutamine cataplerotic efflux and not glycolysis in cells of Cluster 4. In silico analyses of loss-of-function and drug sensitivity screens showed that Cluster 4 cells were more susceptible to gene deletion and drug targeting of lactate and glutamine metabolism, and OXPHOS than cells in Cluster 3. Our results highlight the potential of pathway-centric approaches to reveal new aspects of cellular metabolism from metabolomic data.

cancer biology↗

bioRxiv2022

Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

The unfolding of ubiquitylated proteins by the p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 is essential in many areas of eukaryotic cell biology. Previous studies showed that yeast Cdc48-Ufd1-Npl4 is governed by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that substrate processing by mammalian p97-UFD1-NPL4 involves a complex interplay between ubiquitin chain length and additional p97 cofactors. Using disassembly of the ubiquitylated CMG helicase as a model in vitro system, we find that reconstituted p97-UFD1-NPL4 only unfolds substrates with very long ubiquitin chains. However, this high ubiquitin threshold is greatly reduced, to a level resembling yeast Cdc48-Ufd1-Npl4, by the UBXN7, FAF1 or FAF2 partners of mammalian p97-UFD1-NPL4. Stimulation by UBXN7/FAF1/FAF2 requires the UBX domain that connects each factor to p97, together with the ubiquitin-binding UBA domain of UBXN7 and a previously uncharacterised coiled-coil domain in FAF1/FAF2. Furthermore, we show that deletion of the UBXN7 and FAF1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

molecular biology↗

bioRxiv2016

Development of prokaryotic cell-free systems for synthetic biology

Prokaryotic cell-free systems are currently heavily used for the production of protein that can be otherwise challenging to produce in cells. However, historically cell-free systems were used to explore natural phenomena before the advent of genetic modification and transformation technology. Recently, synthetic biology has seen a resurgence of this historical use of cell-free systems as a prototyping tool of synthetic and natural genetic circuits. For these cell-free systems to be effective prototyping tools, an understanding of cell-free system mechanics must be established that is not purely protein-expression driven. Here we discuss the development of E. coli-based cell-free systems, with an emphasis on documenting published extract and energy preparation methods into a uniform format. We also discuss additional considerations when applying cell-free systems to synthetic biology.

Synthetic Biology↗

bioRxiv2025

MYC/MAX balance dictates cell progenitor fate by altering the HOX program in the Drosophila eye

The transcription factor MYC acts canonically as heterodimer with MAX to promote growth, proliferation, and metabolic adaptation. Although MYC and MAX relative levels are normally kept in balance under physiological conditions, those levels are often dysregulated in many human cancers where MYC is overexpressed and MAX can sometimes be reduced. How altered stoichiometry of MYC/MAX can impact on cell biology is not completely understood. Using the Drosophila eye amenable to genetic modifications, we demonstrate that the relative abundance of the fly MYC ortholog dMyc and its partner dMax affect progenitor cell fate. Elevating the dMyc/dMax ratio--through dMyc or human MYCN overexpression, dMax depletion, or combined manipulations--disrupts photoreceptor differentiation and ectopically induces the thoracic HOX gene Antennapedia within the eye disc. When such a ratio is driven to an extreme degree, the change in cell fate results in eye-to-wing transdetermination. Conversely, restoring dMax levels rescues these phenotypes, demonstrating that stoichiometry, not absolute MYC levels, shapes the response. Transcriptomic profiling reveals global repression of eye-specific regulators (e.g., eyegone and prospero) and ectopic activation of wing determinants (e.g., vestigial and nubbin) while genetic interactions identify the cephalic HOX gene Deformed as a key mediator modulating MYC-dependent identity changes. Our findings uncover MYC/MAX imbalance as a developmental switch that can rewire HOX expression and reprogram progenitor identity, suggesting a conserved mechanism underlying MYC-driven cellular plasticity in cancer.

developmental biology↗