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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 271 records · Page 15Linked to original sources

bioRxiv2014

The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite own actomyosin system and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility.

Cell Biology↗

bioRxiv2014

Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling

Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca2+ waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that \"trigger zones\" with a 60-100 junctions are required to confer a signal of similar magnitude. This is compatible with the 130 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca2+ signal coupling and may contribute to cellular function in health and disease.

Cell Biology↗

bioRxiv2014

Methods to study toxic transgenes in C. elegans: an analysis of protease-dead separase in the C. elegans embryo

We investigated whether the protease activity of separase, which is required for chromosome segregation, is also required for its other roles during anaphase in C. elegans given that non-proteolytic functions of separase have been identified in other organisms. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. The most common method for embryonic expression involves generation of integrated transgenes under the control of the pie-1 promoter, using unc-119 as a selection marker. However expression of dominant-negative proteins kills the strain preventing analysis of mutants. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes in order to study protease-dead separase in embryos. The first involves feeding gfp RNAi to eliminate transgene expression and allows propagation of transgenic lines indefinitely. Animals removed from gfp RNAi for several generations recover transgene expression and associated phenotypes. The second involves propagation of the transgene with the female specific pie-1 promoter via the male germline and analysis of phenotypes in embryos from F1 heterozygous hermaphrodites that express the protein. Using these methods, we show that protease-dead separase causes chromosome nondisjunction and cytokinesis failures. These methods are immediately applicable for studies of dominant-negative transgenes and should open new lines of investigation in the C. elegans embryo.

Cell Biology↗

bioRxiv2014

Non-specificity of Pitstop 2 in clathrin-mediated endocytosis

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. \"Pitstop\" compounds have been developed which block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrins N-terminal domain.

Cell Biology↗

bioRxiv2014

Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4 and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research.

Cell Biology↗

bioRxiv2014

TCF7L2 is a master regulator of insulin production and processing

Although variants in the T-cell factor 7-like 2 gene (TCF7L2) confer the strongest risk of type 2 diabetes (T2D) by presumed effects on islet function, the underlying mechanisms are not well understood. We have identified TCF7L2-target genes and described the regulatory network downstream of TCF7L2 responsible for its effect on insulin secretion in rodents and human pancreatic islets. ISL1 is a direct target of TCF7L2 and regulates proinsulin production and processing via MAFA, PDX1, NKX6.1, PCSK1 and PCSK2 and possibly clearance of proinsulin via SLC30A8. Taken together, these results demonstrate that not only synthesis of proinsulin is regulated by TCF7L2, but also processing and possibly clearance of proinsulin and insulin in a genotype dependent manner. These multiple targets in key pathways may explain why TCF7L2 has emerged as the gene showing the strongest association with T2D.

Cell Biology↗

bioRxiv2014

The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH

Activation of the NADPH oxidase (NOX2) of the phagocytic vacuole of neutrophils is essential for innate immunity. Sustained activity of the oxidase requires that charge movements across the membrane are balanced. A role for the proton channel, HVCN1, has been proposed but not proven. Using the ratiometric pH indicator SNARF, introduced into the cytosol and separately into the vacuole coupled to Candida, we used confocal microscopy to measure changes in pH in these two compartments in human and mouse neutrophils. Shortly after phagocytosis by human cells, the vacuolar pH rose to ~9, at which it was maintained for ~20 minutes, while the cytosol showed a small acidification of ~0.25 pH unit. Alkalinisation has important consequences for the microbicidal and digestive functions of vacuolar enzymes. In HVCN1 knock out mouse neutrophils, the phagocytosis induced respiratory burst was halved to ~3 fmols per Candida, the vacuolar pH rose to >11 and the cytosol acidified excessively to pH ~6.75. These changes were prevented by the protonophore CCCP. The rate of extrusion of protons into the extracellular medium following phagocytosis was not significantly different from wild type neutrophils suggesting that cytoplasmic acidification resulted from the loss of the proton sink into the vacuole. HVCN1 phagocytic vacuoles showed considerable swelling, and this was blocked by CCCP and decreased by valinomycin. Stoichiometric considerations indicated that the HVCN1 channel compensates 90-95% of the oxidase-induced charge in normal cells, and in its absence, charge is carried by ions other than protons, including K+.

Cell Biology↗

bioRxiv2014

MicroRNAs Regulate the Wnt/Ca2+ Signaling Pathway to Promote the Secretion of Insulin in Pancreatic Nestin-Positive Progenitor Cells

MicroRNAs (miRNAs) are small noncoding RNAs that bind to the 3'-UTR of mRNAs and function mainly in post-transcriptional regulation. MiRNAs have been implicated to play roles in organ development, including that of the pancreas. Many miRNAs, such as miR-375, miR-124, miR-7, miR-21 and miR-221, have been shown to regulate insulin production as well as insulin secretion. However, it is not known whether miRNAs can regulate insulin secretion via the control of intracellular Ca2+ in pancreatic beta cells. In this research, expression profiles of miRNAs and mRNAs were investigated using RNA-sequencing and microarray analysis in chicken pancreatic nestin-positive progenitor cells and differentiated pancreatic beta cells. A number of miRNAs were up-regulated after differentiation of progenitors into beta cells, which regulate cell signaling pathways to control cell function. miR-223 and miR146a were shown to promote insulin secretion from pancreatic beta cells by regulating the concentration of intracellular Ca2+ via the down-regulation of their target genes.

Cell Biology↗

bioRxiv2014

Artificially inducing close apposition of endoplasmic reticulum and mitochondria induces mitochondrial fragmentation.

Cycles of mitochondrial fission and fission are essential for normal cell physiology. Defects in the machinery controlling these processes lead to neurodegenerative disease. While we are beginning to understand the machinery that drives fission, our knowledge of the spatial and temporal control of this event is lacking. Here we use a rapamycin-inducible heterodimerization system comprising both ER and mitochondrial transmembrane components to bring the ER membrane into close physical proximity with mitochondria. We show that this artificial apposition of membranes is sufficient to cause rapid mitochondrial fragmentation. Resulting mitochondrial fragments are shown to be distinct entities using fluorescence recovery after photobleaching. We also show that these fragments retain a mitochondrial membrane potential. In contrast, inducible tethering of the peripheral ER exit site protein TFG does not cause mitochondrial fragmentation suggesting that very close apposition of the two membranes is required.

Cell Biology↗

bioRxiv2014

Using Polysome Isolation with Mechanism Alteration to Uncover Transcriptional and Translational Dynamics in Key Genes

What does it mean when we say a cells biochemistry is regulated during changes to the phenotype? While there are a plethora of potential mechanisms and contributions to the final outcome, a more tractable approach is to examine the dynamics of mRNA. This way, we can assess the contributions of both known and unknown decay and aggregation processes for maintaining levels of gene product on a gene-by-gene basis. In this extended protocol, drug treatments that target specific cellular functions (termed mechanism disruption) can be used in tandem with mRNA extraction from the polysome to look at the dynamics of mRNA levels associated with transcription and translation at multiple stages during a physiological perturbation. This is accomplished through validating the polysome isolation method in human cells and comparing fractions of mRNA for each experimental treatment at multiple points in time. First, three different drug treatments corresponding to the arrest of various cellular processes are administered to populations of human cells. For each treatment, the transcriptome and translatome are compared directly at different time points by assaying both cell-type specific and non-specific genes. There are two findings of note. First, extraction of mRNA from the polysome and comparison with the transcriptome can yield interesting information about the regulation of cellular mRNA during a functional challenge to the cell. In addition, the conventional application of such drugs to assess mRNA decay is an incomplete picture of how severely challenged or senescent cells regulate mRNA in response. This extended protocol demonstrates how the gene- and process-variable degradation of mRNA might ultimately require investigations into the course-grained dynamics of cellular mRNA, from transcription to ribosome.

Cell Biology↗

bioRxiv2014

Products of the Parkinson’s-disease related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes, DJ- 1/PARK7, linked with the onset of Parkinson's disease, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

Cell Biology↗

bioRxiv2014

Massively calcified endosomal death (MCED) of endothelial cells

We have discovered a novel and specific mechanism of endothelial cell death. We refer to this novel death mechanism as massively calcified endosomal death, or MCED. Exposure of endothelial cells to non-specific toxins or other physical stresses induces death by traditional apoptotic and non-apoptotic mechanisms, common to most different types of cells. In contrast, exposure of endothelial cells (but not other types of nucleated cells) to specific insults, such as oxidized pathogenic lipids (e.g. 7-ketocholesterol) or agents with known anti-angiogenic activity (e.g. bevacizumab, certain tyrosine kinase inhibitors, etc.) triggers cell death via a novel pathway, which involves the formation of massively calcified endosomes, which, in turn, escape from the dying endothelial cells as massively calcified exosomes. These endosomes/exosomes appear capable of provoking an inflammatory response, characterized by physical association of calcified microparticles with inflammatory cells (monocytes, lymphocytes, neutrophils) with resulting increased release of an inflammatory mediator (TNF) into the culture medium. Traditional media for the culture of endothelial cells are profoundly inhibitory to MCED, as are some mammalian sera and many human sera, explaining why MCED had not been previously discovered and reported. The present discovery of MCED was accidental, resulting from work with primary cultures of fresh human tumor cell clusters, which invariably contain microcapillary cells. Our culture media are optimized for the tumor cells and not for the endothelial cells and, thus, are permissive of MCED. I propose MCED as the central mechanism underlying both intimal calcification and vascular inflammation in atherosclerosis.

Cell Biology↗

bioRxiv2014

TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation

The interaction between TACC3 (transforming acidic coiled coil protein 3) and the microtubule polymerase ch-TOG (colonic, hepatic tumor overexpressed gene) is evolutionarily conserved. Loading of TACC3-ch-TOG onto mitotic spindle microtubules requires the phosphorylation of TACC3 by Aurora-A kinase and the subsequent interaction of TACC3 with clathrin to form a microtubule-binding surface. Recent work indicates that TACC3 can track the plus-ends of microtubules and modulate microtubule dynamics in non-dividing cells via its interaction with ch-TOG. Whether there is a pool of TACC3-ch-TOG that is independent of clathrin in human cells, and what is the function of this pool, are open questions. Here, we describe the molecular interaction between TACC3 and ch-TOG that permits TACC3 recruitment to the plus-ends of microtubules. This TACC3-ch-TOG pool is independent of EB1, EB3, Aurora-A phosphorylation and binding to clathrin. We also describe the distinct combinatorial subcellular pools of TACC3, ch-TOG and clathrin. TACC3 is often described as a centrosomal protein, but we show that there is no significant population of TACC3 at centrosomes. The delineation of distinct protein pools reveals a simplified view of how these proteins are organized and controlled by post-translational modification.

Cell Biology↗

bioRxiv2015

Mbd3 and deterministic reprogramming

Embryonic development requires the activity of the Nucleosome Remodeling and Deacetylase (NuRD) complex. NuRD functionality can be ablated by rendering cells devoid of methyl-CpG-binding domain protein 3 (Mbd3), a critical component that confers stability to the complex1. Previous studies noted that Mbd3-/- embryonic stem (ES) cells misregulate a subset of pluripotency-associated genes, and subsequently fail to engage in cell differentiation into embryonic lineages when self-renewal requisites (e.g. LIF) are withdrawn from culture media2-3. Components of the NuRD complex have been shown to interact with Oct4 and Nanog, two important transcription factors operative in the production of iPS cells4-7. Thus, elucidating the role of Mbd3/NuRD in the reprogramming process is of relevance to the field.\n\nRais et al. reported the remarkable obse ...

Cell Biology↗

bioRxiv2015

Ca-activation kinetics modulate successive puff/spark amplitude, duration and inter-event-interval correlations in a Langevin model of stochastic Ca release

Through theoretical analysis of the statistics of stochastic calcium (Ca2+) release (i.e., the amplitude, duration and inter-event interval of simulated Ca2+ puffs and sparks), we show that a Langevin description of the collective gating of Ca2+ channels may be a good approximation to the corresponding Markov chain model when the number of Ca2+ channels per Ca2+ release unit (CaRU) is in the physiological range. The Langevin description of stochastic Ca2+ release facilitates our investigation of correlations between successive puff/spark amplitudes, durations and inter-spark intervals, and how such puff/spark statistics depend on the number of channels per release site and the kinetics of Ca2+-mediated inactivation of open channels. When Ca2+ inactivation/de-inactivation rates are intermediate--i.e., the termination of Ca2+ puff/sparks is caused by the recruitment of inactivated channels--the correlation between successive puff/spark amplitudes is negative, while the correlations between puff/spark amplitudes and the duration of the preceding or subsequent inter-spark interval are positive. These correlations are significantly reduced when inactivation/deinactivation rates are extreme (slow or fast) and puff/sparks terminate via stochastic attrition.

Cell Biology↗

bioRxiv2015

Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via post-translational modification of its targets with polyubiquitin chains. These commonly contain Lysine 48 (K48)-directed ubiquitin linkages, but chains containing atypical Lysine 11 (K11) linkages also target substrates to the proteasome, for example to regulate cell cycle progression. A single ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC/C), controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We have tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single cell level. We report that all anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, while other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a co-activator-specified manner.\n\nAbbreviations

Cell Biology↗

bioRxiv2015

Engineering Cell Fate: The Roles of iPSC Transcription Factors, Chemicals, Barriers and Enhancing Factors in Reprogramming and Transdifferentiation

Direct reprogramming technology has emerged as an outstanding technique for the generation of induced pluripotent stem (iPS) cells and various specialized cells directly from somatic cells of different species. Recent studies dissecting the molecular mechanisms of reprogramming have methodologically improved the quality, ease and efficiency of reprogramming and eliminated the need for genome modifications with integrating viral vectors. With these advancements, direct reprogramming technology has moved closer to clinical application. Here, we provide a comprehensive overview of the cutting-edge findings regarding distinct barriers of reprogramming to pluripotency, strategies to enhance reprogramming efficiency, and chemical reprogramming as one of the non-integrating approaches in iPS cell generation. In addition to direct transdifferentiation, pluripotency factor-induced transdifferentiation or cell activation and signaling directed (CASD) lineage conversion is described as a robust strategy for the generation of both tissue-specific progenitors and clinically relevant cell types. Then, we consider the possibility that a combined method of inhibition of roadblocks (e.g. p53, p21, p57, Mbd3, etc.), and application of enhancing factors in a chemical reprogramming paradigm would be an almost safe, reliable and effective approach in pluripotent reprogramming and transdifferentiation. Furthermore, with respect to the state of native, aberrant, and target gene regulatory networks in reprogrammed cell populations, CellNet is reviewed as a computational platform capable of evaluating the fidelity of reprogramming methods and refining current engineering strategies. Ultimately, we conclude that a faithful, highly efficient and integration-free reprogramming paradigm would provide powerful tools for research studies, drug-based induced regeneration, cell transplantation therapies and other regenerative medicine purposes.

Cell Biology↗

bioRxiv2015

Myc-induced cell mixing is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan1. There is no clear evidence that intercalation is involved in pathologies. Here, we use the proto-oncogene Myc to study a process analogous to early phase of tumour expansion: Myc-induced cell competition2-7. Cell competition is a conserved mechanism5,6,8,9 driving the elimination of slow proliferating cells (so called losers) by faster proliferating neighbours (so called winners) through apoptosis10 and is important to prevent developmental malformations and maintain tissue fitness11. Using long term live imaging of Myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, we show that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser/winner interface morphology can modulate the strength of competition. We further show that elimination of loser clones requires winner/loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilisation of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference of junctional F-actin levels between loser and winner junctions, induced by differential levels of the phosphatidylinositol PIP3. Our results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues.

Cell Biology↗