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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 235 records · Page 13Linked to original sources

bioRxiv2018

Lysophosphatidic acid provokes fibroblast chemotaxis through combinatorial regulation of myosin II

Lysophophatidic acid (LPA), a biologically active phospholipid that is ubiquitously present in tissues and organs, provokes cellular responses such as proliferation, apoptosis, differentiation and migration via activation of G-protein coupled receptors. These receptors activate a broad range of intracellular signaling cascades to mediate these responses. Using microfluidic chambers that generate and maintain stable gradients, we observed that chemotaxis of fibroblasts to LPA has higher directional fidelity than chemotaxis provoked by the receptor tyrosine kinase (RTK) ligand platelet-derived growth factor (PDGF). Unlike fast moving amoeboid cells, mesenchymal cells such as fibroblasts do not require PI3K for chemotaxis to a GPCR ligand. In addition, the Arp2/3 complex is not required for fibroblast GPCR-based chemotaxis in either 2D or 3D environments. Our data indicate that combinatorial regulation of myosin II involving global activation by RhoA/ROCK and local inhibition of myosin II at the leading edge by PKC results in highly efficient chemotaxis of fibroblasts to LPA. Based on these observations, we develop a simple mathematical model to explain how dual regulation of myosin II is responsible for enhanced chemotaxis in LPA gradients relative to PDGF. Using pharmacological approaches, we test predictions of this model and modulate the fidelity of LPA and PDGF chemotaxis.

cell biology↗

bioRxiv2017

A genome-wide interactome of DNA-associated proteins in the human liver

Large-scale efforts like the Encyclopedia of DNA Elements (ENCODE) Project have made tremendous progress in cataloging the genomic binding patterns of DNA-associated proteins (DAPs), such as transcription factors (TFs). However most chromatin immunoprecipitation-sequencing (ChIP-seq) analyses have focused on a few immortalized cell lines whose activities and physiology deviate in important ways from endogenous cells and tissues. Consequently, binding data from primary human tissue are essential to improving our understanding of in vivo gene regulation. Here we analyze ChIP-seq data for 20 DAPs assayed in two healthy human liver tissue samples, identifying more than 450,000 binding sites. We integrated binding data with transcriptome and phased whole genome data to investigate allelic DAP interactions and the impact of heterozygous sequence variation on the expression of neighboring genes. We find our tissue-based dataset demonstrates binding patterns more consistent with liver biology than cell lines, and describe uses of these data to better prioritize impactful non-coding variation. Collectively, our rich dataset offers novel insights into genome function in healthy liver tissue and provides a valuable research resource for assessing disease-related disruptions.

genomics↗

bioRxiv2017

Extensive alternative splicing transitions during postnatal skeletal muscle development are required for Ca2+ handling

Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first two weeks after birth are particularly dynamic for differential gene expression and AS transitions, and calciumhandling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin targets Nfatc3 and Nfatc2, and affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of AS during muscle postnatal development.

cell biology↗

bioRxiv2018

Continuous visualization of differences between biological conditions in single-cell data

In high-dimensional single cell data, comparing changes in functional markers between conditions is typically done across manual or algorithm-derived partitions based on population-defining markers. Visualizations of these partitions is commonly done on low-dimensional embeddings (eg. t-SNE), colored by per-partition changes. Here, we provide an analysis and visualization tool that performs these comparisons across overlapping k-nearest neighbor (KNN) groupings. This allows one to color low-dimensional embeddings by marker changes without hard boundaries imposed by partitioning. We devised an objective optimization of k based on minimizing functional marker KNN imputation error. Proof-of-concept work visualized the exact location of an IL-7 responsive subset in a B cell developmental trajectory on a t-SNE map independent of clustering. Per-condition cell frequency analysis revealed that KNN is sensitive to detecting artifacts due to marker shift, and therefore can also be valuable in a quality control pipeline. Overall, we found that KNN groupings lead to useful multiple condition visualizations and efficiently extract a large amount of information from mass cytometry data. Our software is publicly available through the Bioconductor package Sconify.

systems biology↗

bioRxiv2017

Two spatially distinct Kinesin-14 Pkl1 and Klp2 generate collaborative inward forces against Kinesin-5 Cut7 in S. pombe

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by Kinesin-5 that generates outward force. This outward force is balanced by antagonistic inward force elicited by Kinesin-14 and/or Dynein. In fission yeast, two Kinesin-14s, Pkl1 and Klp2, play an opposing role against Kinesin-5/Cut7. However, how these two Kinesin-14s coordinate individual activities remains elusive. Here we show that while deletion of either pkl1 or klp2 rescues temperature sensitive cut7 mutants, only pkl1 deletion can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, while Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two Kinesin-14s. Interestingly, human Kinesin-14/HSET can replace either Pkl1 or Klp2. Moreover, overproducing HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism of how two different Kinesin-14s exert their antagonistic roles against Kinesin-5 in a spatially distinct manner.\n\nSUMMARY STATEMENTProper force-balance generated by Kinesin-5 and Kinesin-14 is crucial for spindle bipolarity. Two fission yeast Kinesin-14s localize to different structures, thereby collaboratively producing inward forces against Kinesin-5-mediated outward force.\n\nAbbreviations used

cell biology↗

bioRxiv2018

Multicolor single particle reconstruction of protein complexes

Single-particle reconstruction (SPR) from electron microscopy images is widely used in structural biology, but lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and co-aligns multiple proteins from 2D super-resolution fluorescence images. We demonstrate our method by generating multi-color 3D reconstructions of several proteins within the human centriole and procentriole, revealing their relative locations, dimensions and orientations.

cell biology↗

bioRxiv2017

Inferring intracellular signal transduction circuitry from molecular perturbation experiments

The development of network inference methodologies that accurately predict connectivity in dysregulated pathways may enable the rational selection of patient therapies. Accurately inferring an intracellular network from data remains a very challenging problem in molecular systems biology. Living cells integrate extremely robust circuits that exhibit significant heterogeneity, but still respond to external stimuli in predictable ways. This phenomenon allows us to introduce a network inference methodology that integrates measurements of protein activation from perturbation experiments. The methodology relies on logic-based networks to provide a predictive approximation of the transfer of signals in a network. The approach presented was validated in silico with a set of test networks and applied to investigate the epidermal growth factor receptor signaling of a breast epithelial cell line, MFC10A. In our analysis, we predict the potential signaling circuitry most likely responsible for the experimental readouts of several proteins in the mitogen activated protein kinase and phosphatidylinositol-3 kinase pathways. The approach can also be used to identify additional necessary perturbation experiments to distinguish between a set of possible candidate networks.

systems biology↗

bioRxiv2018

Systematic assessment of GFP tag position on protein localization and growth fitness in yeast

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on localization and function would promote accurate use of tags in experimental setups. Here we examine the effect of Green Fluorescent Protein (GFP) tagging at either the N or C terminus of budding yeast proteins on localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides step towards broad investigation of protein fusion libraries.\n\nHighlightsO_LIProtein tags are widely used in molecular biology although they may interfere with protein function.\nC_LIO_LIThe subcellular localization of hundreds of proteins in yeast is different when tagged at the N or the C terminus.\nC_LIO_LIA competition based assay enables systematic deciphering of correct tagging terminus for essential proteins.\nC_LIO_LIThe presented approach can be used to derive physiologically relevant tagged libraries.\nC_LI

cell biology↗

bioRxiv2017

Enzymatic production of single molecule FISH and RNA capture probes

Arrays of singly-labelled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single molecule microscopy analysis and high efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that 1) all steps of the oligonucleotide labelling - including conjugation, enzymatic synthesis and product purification - can be performed in a standard biology laboratory, 2) the process yields >90 %, often >95 % labeled product with minimal carry-over of impurities and 3) the oligonucleotides can be labeled with different dyes or biotin, allowing single molecule FISH or RNA affinity purification to be performed.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC=\"FIGDIR/small/107599_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@a26162org.highwire.dtl.DTLVardef@a780d9org.highwire.dtl.DTLVardef@fc3a62org.highwire.dtl.DTLVardef@15d183c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Quantitative Real-Time Imaging of Glutathione with Sub-Cellular Resolution

Quantitative imaging of glutathione with high spatial and temporal resolution is essential for studying the roles of glutathione in redox biology. We developed a fluorescent glutathione probe--HaloRT--that targets organelles of interest through expressing organelle-specific HaloTag proteins. Using HaloRT, we quantitatively measure the glutathione concentrations in the nucleus and the cytosol and find no appreciable concentration gradient between these two organelles, challenging the view of nuclear compartmentalization of glutathione.

cell biology↗

bioRxiv2018

Multiplexed imaging using same species primary antibodies with signal amplification

Immunofluorescence (IF) imaging using antibodies to visualize specific biomolecules is a widely used technique in both biological and clinical laboratories. Standard IF imaging methods using primary antibodies followed by secondary antibodies have low multiplexing capability due to limited availability of primary antibodies raised in different animal species. Here, we used a DNA-based signal amplification method, Hybridization Chain Reaction (HCR), to replace secondary antibodies to achieve multiplexed imaging using primary antibodies of the same species with superior signal intensity. To enable imaging with DNA-conjugated antibodies, we developed a new antibody staining protocol to minimize nonspecific binding of antibodies caused by conjugated DNA oligonucleotides. We also expanded the HCR hairpin pool from previously published 5 to 13 for highly multiplexed in situ imaging. We finally demonstrated multiplexed in situ protein imaging using the technique in both cultured cells and mouse retina sections.

cell biology↗

bioRxiv2018

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

cell biology↗

bioRxiv2018

Three-photon light-sheet fluorescence microscopy

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 {micro}m diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

cell biology↗

bioRxiv2026

Functional characterization of Rho GTPase activating proteins SYDE1 and SYDE2

The human genome encodes more than 60 proteins containing Rho GTPase activating protein (RhoGAP) domains, many of which remain understudied with respect to their target specificity and biological roles. SYDE1 and SYDE2 are two such orphan RhoGAPs, for which there are few studies characterizing their biochemical and cellular functions and conflicting reports identifying their cognate GTPases. We previously identified SYDE1 and SYDE2 in a screen for substrates of the c-Jun N-terminal kinases. Here, we show that SYDE1 and SYDE2 are preferentially phosphorylated by JNK1 relative to other mitogen-activated protein kinases (MAPKs) at sites proximal to a kinase docking region. Purified SYDE1 and SYDE2 are shown to have significant catalytic GAP activity toward RhoA, Rac1, and Cdc42. However, neither up- nor down-regulation of SYDE1/2 expression leads to detectable changes in bulk GTP loading of any of these GTPases. Nevertheless, we demonstrate that SYDE1 and SYDE2, in a partially GAP-dependent manner, increase cell spreading and number of focal adhesions, and promote more directionally persistent migration in HEK293 cells. Together, these findings establish SYDE1 and SYDE2 as robust JNK substrates with catalytic activity toward a set of Rho GTPases and reveal basic functions of SYDE1 and SYDE2 in regulating cell morphology, adhesion, and migration.

cell biology↗

bioRxiv2018

CRISPR/Cas9 gene editing to make conditional mutants of the human malaria parasite Plasmodium falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of better drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology, but historically, the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. The method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

cell biology↗

bioRxiv2017

Airway IRF7hi versus IRF7lo molecular response patterns determine clinical phenotypes in children with acute wheezing

Asthma exacerbations are triggered by rhinovirus infections. We employed a systems biology approach to delineate upper airway gene network patterns underlying asthma exacerbation phenotypes in children. Cluster analysis unveiled distinct IRF7hi versus IRF7lo molecular phenotypes, the former exhibiting robust upregulation of Th1/type I interferon responses and the latter an alternative signature marked by upregulation of cytokine and growth factor signalling and downregulation of interferon gamma. The two phenotypes also produced distinct clinical phenotypes. For IRF7lo versus IRF7hi: symptom duration prior to hospital presentation was more than twice as long from initial symptoms (p=0.011) and nearly three times as long for cough (p<0.001); the odds ratio of admission to hospital was increased more than four-fold (p=0.018); and time to recurrence was shorter (p=0.015). In summary, our findings demonstrate that asthma exacerbations in children can be divided into IRF7hi versus IRF7lo phenotypes with associated differences in clinical phenotypes.\n\nAbbreviationsAHR, airway hyperresponsiveness; ARG1, Arginase 1, CSF3, Colony Stimulating Factor 3; CD38, Cluster of Differentiation 38; CD163, Cluster of Differentiation 163; cDCs, conventional (or myeloid) dendritic cells; DDX60, DExD/H-Box Helicase 60; ED, Emergency Department; EGF, Epidermal Growth Factor; ERK, Extracellular signal-Regulated Kinase; FCER1G, Fc Fragment Of IgE Receptor Ig; HMBS, Hydroxymethylbilane Synthase; IFNg, Interferon Gamma; IFNL1, Interferon Lambda 1; IL-1R2, Interleukin 1 Receptor Type 2; IRF7, Interferon Regulatory Factor 7; ISG15, Interferon-stimulated gene 15; MDA5, Melanoma Differentiation-Associated protein 5; MX1, Myxovirus Resistance Protein 1; NAD, nicotinamide adenine dinucleotide; NCR1, Natural cytotoxicity triggering receptor 1; OSM, Oncostatin M; PD-L1, Programmed Death-Ligand 1; PPIA, Peptidylprolyl Isomerase A; PPIB Peptidylprolyl Isomerase B; RSAD2, Radical S-adenosyl methionine domain-containing protein 2; RSV, respiratory syncytial virus; RT-qPCR, quantitative reverse transcription PCR; RV, rhinovirus; sPLA2, secretory Phospholipase A2; TGFb, Transforming Growth Factor beta; THBS1, Thrombospondin 1; TNF, Tumor Necrosis Factor; TLR2, Toll-like Receptor 2.

cell biology↗

bioRxiv2018

CDeep3M - Plug-and-Play cloud based deep learning for image segmentation of light, electron and X-ray microscopy

As biological imaging datasets increase in size, deep neural networks are considered vital tools for efficient image segmentation. While a number of different network architectures have been developed for segmenting even the most challenging biological images, community access is still limited by the difficulty of setting up complex computational environments and processing pipelines, and the availability of compute resources. Here, we address these bottlenecks, providing a ready-to-use image segmentation solution for any lab, with a pre-configured, publicly available, cloud-based deep convolutional neural network on Amazon Web Services (AWS). We provide simple instructions for training and applying CDeep3M for segmentation of large and complex 2D and 3D microscopy datasets of diverse biomedical imaging modalities.

cell biology↗

bioRxiv2017

High contrast staining for serial block face scanning electron microscopy without uranyl acetate

1Serial block face scanning electron microscopy (SBFSEM) is an increasingly popular method for investigating the three-dimensional ultrastructure of large biological samples. Prior to imaging, samples are typically chemically fixed, stained with osmium and uranyl acetate, and subsequently embedded in resin. The purpose of staining is to provide image contrast and reduce specimen charging under the electron beam, which is detrimental to the quality of imaging. Obtaining, using, and disposing of uranyl acetate is getting increasingly cumbersome in many countries due to new regulations on the handling of radioactive substances. Therefore, we developed an alternative staining procedure that does not rely on the use of uranium or any other radioactive substance. This procedure provides excellent contrast and efficiently reduces specimen charging.

cell biology↗