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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 145 records · Page 8Linked to original sources

bioRxiv2017

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. Almost nothing is known about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25s endogenous RNA targets and protein binding partners. Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase function. These results reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor for their biological functions.

molecular biology↗

bioRxiv2017

FACSanadu: Graphical user interface for rapid visualization and quantification of flow cytometry data

MotivationFlow cytometry is a fundamental technique in cell biology, yet few open source packages are available to analyse these data. Here we describe FACSanadu, an interactive package for rapid visualization and measurement of flow cytometry data. It is the first open source package that can read length profile data from the COPAS Biosorter.\n\nAvailability and ImplementationFACSanadu is implemented in Java and uses the Qt framework for display. Binary distributions are made for all major operating systems (Windows, Macintosh, Linux). The source code and documentation is available as free software at http://www.facsanadu.org.\n\nContact: mahogny@areta.org

bioinformatics↗

bioRxiv2017

An atlas of genetic variation for linking pathogen-induced cellular traits to human disease

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with disease. To facilitate moving from associations to disease mechanisms, we leveraged the role of pathogens in shaping human evolution with the Hi-HOST Phenome Project (H2P2): a catalog of cellular GWAS comprised of 79 phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance (p<5x10-8) for phenotypes ranging from pathogen replication to cytokine production. Combining H2P2 with clinical association data from the eMERGE Network and experimental validation revealed evidence for mechanisms of action and connections with diseases. We identified a SNP near CXCL10 as a cis-cytokine-QTL and a new risk factor for inflammatory bowel disease. A SNP in ZBTB20 demonstrated pleiotropy, partially mediated through NF{kappa}B signaling, and was associated with viral hepatitis. Data are available in an H2P2 web portal to facilitate further interpreting human genome variation through the lens of cell biology.

genetics↗

bioRxiv2017

A multi-center study on factors influencing the reproducibility of in vitro drug-response studies

Evidence that some influential biomedical results cannot be repeated has increased interest in practices that generate data meeting findable, accessible, interoperable and reproducible (FAIR) standards. Multiple papers have identified examples of irreproducibility, but practical steps for increasing reproducibility have not been widely studied. Here, seven research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cell biology, and patient stratification. While many experimental and computational factors have an impact on intra- and inter-center reproducibility, the factors most difficult to identify and correct are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways of identifying such context-sensitive factors, thereby advancing the conceptual and practical basis for greater experimental reproducibility.

cancer biology↗

bioRxiv2017

Promoter boundaries for the luxCDABE and betIBA-proXWV operons in Vibrio harveyi defined by the method RAIL: Rapid Arbitrary PCR Insertion Libraries

Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi. We describe a new method called RAIL (Rapid Arbitrary PCR Insertion Libraries) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of the luxCDABE and betIBA-proXWV operons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method to gfp, mCherry, and lacZ reporters and multiple promoters in V. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.\n\nImportanceGene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacterium Vibrio harveyi by constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.

molecular biology↗

bioRxiv2017

RNA sequencing of Stentor cell fragments reveals key processes underlying cellular regeneration

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 up-regulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many up-regulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes such as dual-specific tyrosine-(Y)-phosphorylation regulated kinases and MORN domain containing proteins that seemingly act as key-regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies, our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

genomics↗

bioRxiv2018

Quantitative variation within a species for traits underpinning C4 photosynthesis

Engineering C4 photosynthesis into C3 crops such as rice or wheat could substantially increase their yield by alleviating photorespiratory losses1,2. This objective is challenging because the C4 pathway involves complex modifications to the biochemistry, cell biology and anatomy of leaves3. Forward genetics has provided limited insight into the mechanistic basis of these characteristics and there have been no reports of significant quantitative intra-specific variation of C4 attributes that would allow trait-mapping4,5. Here we show that accessions of C4 Gynandropsis gynandra collected from locations across Africa and Asia exhibit natural variation in key characteristics of C4 photosynthesis. Variable traits include bundle sheath size and vein density, gas exchange parameters and carbon-isotope discrimination associated with the C4 state, but also abundance of transcripts encoding core enzymes of the C4 cycle. Traits relating to water use showed more quantitative variation than those associated with carbon assimilation. We propose variation in these traits likely adapted the hydraulic system for increased water use efficiency rather than improving carbon fixation, indicating that selection pressure may drive C4 diversity in G. gynandra by acting to modify water use rather than photosynthesis. As these accessions can be easily crossed and produce fertile offspring, our findings indicate that natural variation within a C4 species is sufficiently large to allow genetic-mapping of key anatomical C4 traits and regulators.

plant biology↗

bioRxiv2018

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein prior to its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data are only available for MurJ from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the Gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

biochemistry↗

bioRxiv2018

Specific Eph Receptor-Cytoplasmic Effector Signaling Mediated by SAM-SAM Domain Interactions

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain numerous Eph SAM mutations identified in patients suffering from cancers and other diseases.

biophysics↗

bioRxiv2018

A Bayesian sequential learning framework to parameterise continuum models of melanoma invasion into human skin

We present a novel framework to parameterise a mathematical model of cell invasion that describes how a population of melanoma cells invades into human skin tissue. Using simple experimental data extracted from complex experimental images, we estimate three model parameters:(i) the melanoma cell proliferation rate,{lambda} ; (ii) the melanoma cell diffusivity, D; and (iii){delta} , a constant that determines the rate that melanoma cells degrade the skin tissue. The Bayesian sequential learning frame-work involves a sequence of increasingly-sophisticated experimental data from:(i) a spatially uniform cell proliferation assay; (ii) a two-dimensional circular barrier assay; and, (iii) a three-dimensional invasion assay. The Bayesian sequential learning approach leads to well-defined parameter estimates. In contrast, taking a naive approach that attempts to estimate all parameters from a single set of images from the same experiment fails to produce meaningful results. Overall our approach to inference is simple-to-implement, computationally efficient, and well-suited for many cell biology phenomena that can be described by low dimensional continuum models using ordinary differential equations and partial differential equations. We anticipate that this Bayesian sequential learning framework will be relevant in other biological contexts where it is challenging to extract detailed, quantitative biological measurements from experimental images and so we must rely on using relatively simple measurements from complex images.

biophysics↗

bioRxiv2018

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

genetics↗

bioRxiv2018

Nemo-like kinase regulates Progranulin levels in the brain through the microglial endocytosis-lysosomal pathway

Genetic variants in Granulin (GRN), which encodes the secreted glycoprotein Progranulin (PGRN), are associated with several neurodegenerative diseases including frontotemporal lobar degeneration, neuronal ceroid lipofuscinosis, and Alzheimers disease. These genetic alterations manifest in pathological changes due to a reduction of PGRN expression; therefore, identifying a factor that can modulate PGRN levels in vivo would enhance our understanding of PGRN in neurodegeneration, and could reveal novel potential therapeutic targets. Here, we report that Nemo-like kinase (Nlk) regulates Pgrn levels and its associated neuropathophysiology. Genetic interaction studies in mice show that Grn heterozygote mice on an Nlk heterozygote background display pathological and behavioral phenotypes which mimic Grn knockout mice. Furthermore, biochemical and cell biological studies suggest that Nlk reduction promotes Pgrn degradation via the endocytosis-lysosomal pathway, specifically in microglia. Our results reveal a new mechanism for the regulation of Pgrn in the brain and provide insight into the pathophysiology of PGRN-associated diseases.

neuroscience↗

bioRxiv2018

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

synthetic biology↗

bioRxiv2018

MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.

neuroscience↗

bioRxiv2018

A nanotube-mediated path to protocell formation

Cellular compartments are membrane-enclosed, spatially distinct microenvironments which confine and protect biochemical reactions in the biological cell. On the early Earth, the autonomous formation of compartments is thought to have led to the encapsulation of nucleotides, thereby satisfying a starting condition for the emergence of life. Recently, surfaces have come into focus as potential platforms for the self-assembly of prebiotic compartments, as significantly enhanced vesicle formation was reported in the presence of solid interfaces. The detailed mechanism of such formation at the mesoscale is still under discussion. We report here on the spontaneous transformation of solid surface-adhered lipid deposits to unilamellar membrane compartments through a straightforward sequence of topological changes, proceeding via a network of interconnected lipid nanotubes. We show that this transformation is entirely driven by surface-free energy minimization and does not require hydrolysis of organic molecules, or external stimuli such as electrical currents or mechanical agitation. The vesicular structures take up and encapsulate their external environment during formation, and can subsequently separate and migrate upon exposure to hydrodynamic flow. This may link, for the first time, the self-directed transition from weakly organized bioamphiphile assemblies on solid surfaces to protocells with secluded internal contents.\n\nSignificanceThe nature of the physical and chemical mechanisms behind the formation, growth and division of the earliest protocells is among the key questions concerning the origin of life. Establishing a simple pathway for the assembly of protocell structures from the primordial soup is a particular challenge. Emerging evidence supporting the assumption that solid surfaces have a governing role in protocell formation has recently expanded the scope, and created new inspiration for investigation. By presenting a physical path from self-assembled amphiphile-based membranes on solid surfaces to spherical single-membrane compartments via a consistent sequence of transformations, solely driven by the materials properties of the interfaces, a direct link between the presence of functional biomolecules and the development of protocells can be established.

biophysics↗

bioRxiv2018

A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this cloning-free approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applies as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.

molecular biology↗

bioRxiv2018

Environment and HSP90 modulate MAPK stomatal developmental pathway

Stomatal ontogenesis is a key element of plant adaptation aiming to control photosynthetic efficiency and water management under fluctuating environments 1,2,3. Development of stomata is guided by endogenous and environmental cues and is tightly coupled to overall plant growth 1,2,3. YODA signaling pathway is essential to stomatal lineage specification4,5,6 since it regulates the activities of transcription factors such as SPEECHLESS (SPCH)7,8,9,10. Heat-shock proteins 90 (HSP90s) are evolutionarily conserved molecular chaperones implicated in a broad range of signalling pathways being integrated in interaction networks with client proteins11,12,13,14. Herein, based on genetic, molecular, biochemical, and cell biological evidence we report that heat-stress conditions affect phosphorylation and deactivation of SPCH and modulate stomatal density. We show that genetic and physical interactions between HSP90s and YODA control stomatal patterning, distribution and morphology. We provide solid evidence that HSP90s play a major role in transducing the heat-stress response since they act upstream and downstream of YODA signalling, regulate the activity and nucleocytoplasmic distribution of MAPKs, and the activation of SPCH. Thus, HSPs control the stomatal development both under normal temperature and acute heat-stress conditions. Our results demonstrate that HSP90s couple stomatal formation and patterning to environmental cues providing an adaptive mechanism of heat-stress tolerance response and stomatal formation in Arabidopsis.

plant biology↗

bioRxiv2018

In vivo assembly and trafficking of olfactory Ionotropic Receptors

lonotropic Receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors(iGluRs), with roles in chemosensation, thermosensation and hygrosensation. Analogous to the synaptic targeting mechanisms of their iGluR ancestors, IRs are thought to form complexes of broadly-expressed co-receptors and selectively-expressed tuning receptors to localise to sensory cilia. While tuning receptors extracellular ligand-binding domain (LBD) defines sensory specificity, the role of this domain in co-receptors is unclear. We identify a coreceptor-specific sequence in the LBD, which contains a single N-glycosylation site. Combining molecular genetic and cell biological analyses, we show that this site is dispensable for assembly of IR complexes in olfactory sensory neurons, but essential for endoplasmic reticulum exit of some,but not all, IR complexes. Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes, and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.

neuroscience↗