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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 307 records · Page 17Linked to original sources

bioRxiv2016

Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials

There is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&E, Massons Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial.

Cell Biology↗

bioRxiv2016

CELL SEGMENTATION WITH RANDOM FERNS AND GRAPH-CUTS

The progress in imaging techniques have allowed the study of various aspect of cellular mechanisms. To isolate individual cells in live imaging data, we introduce an elegant image segmentation framework that effectively extracts cell boundaries, even in the presence of poor edge details. Our approach works in two stages. First, we estimate pixel interior/border/exterior class probabilities using random ferns. Then, we use an energy minimization framework to compute boundaries whose localization is compliant with the pixel class probabilities. We validate our approach on a manually annotated dataset.

Cell Biology↗

bioRxiv2016

Rigidity of silicone substrates controls cell spreading and stem cell differentiation

Multiple functions of cells cultured on flat substrates have been shown to depend on the elastic modulus of the substrate, E, with the dependence being strongest in a physiological range of soft tissues, corresponding to E from 0.1 to 100 kPa. Among those functions are stem cell differentiation, cell spreading, and cell signaling [1]. In the context of differentiation of mesenchymal stem cells (MSCs), substrates with E in the ranges of <4 kPa, 8-17 kPa, and >25 kPa, have been classified as soft (adipogenic) [2,3], medium rigidity (myogenic)1, and hard (osteogenic) [1], respectively. In most studies, the soft substrates are hydrogels, and variations in their elastic moduli are usually accompanied by variations in the dry mass and porosity. The paradigm of the effect of substrate rigidity on the cellular functions has been challenged by Trappmann et al. [4], who claimed that cell spreading and differentiation on hydrogel substrates depend not on the elastic moduli of the substrates, but rather on their porosity, which affects the density of adhesion points between the substrate surface and the extracellular matrix (ECM) coating on it. This claim has been rebutted by Wen at al. [3], who have used hydrogel substrates with different porosities but identical elastic moduli to show that it is the elastic modulus rather than the porosity that is key to the effect of the substrate on cell spreading and differentiation. Both publications agree, however, that there is no appreciable effect of the substrate rigidity on either cell spreading or differentiation, if the substrate is made of a silicone gel rather than a hydrogel. This conclusion appears to contradict the findings of several other groups, who reported that when cells are plated on an array of flexible silicone microposts, their spreading and differentiation depend on the rigidity of the substrate [5], and that when cell are plated on silicone gels, their differentiation depends on the gel rigidity [6]. To resolve this contradiction, we used soft, medium, and hard silicone gel substrates with elastic moduli of 0.5, 16, and 64 kPa, respectively, (Fig.1) to perform experiments similar to those reported in Refs.4 and 3, testing the dependence of differentiation and spreading of MSCs and of spreading of fibroblasts and keratinocytes on the substrate rigidity.

Cell Biology↗

bioRxiv2016

Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

Cas9 endonuclease can be targeted to genomic sequences by varying the sequence of the single guide RNA (sgRNA). The activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines, or using inefficient sgRNAs, can require extensive downstream screening to identify homozygous clones. We have found that linear, non-homologous oligonucleotide DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation greatly increases the frequency of clones with homozygous gene disruptions, even in polyploid cell lines, and rescues otherwise ineffective sgRNAs. The mechanism of enhanced gene disruption differs between human cell lines, stimulating deletion of genomic sequence and/or insertion of non-homologous oligonucleotide DNA at the edited locus in a cell line specific manner. Thus, the addition of non-homologous DNA appears to drive cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

Cell Biology↗

bioRxiv2016

C. elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

Caenorhabditis elegans senses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by the mec-4(d) mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on the Xenopus oocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components.

Cell Biology↗

bioRxiv2016

Distinct stages in the recognition, sorting and packaging of proTGFα into COPII coated transport vesicles

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former employs more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGF, the transmembrane precursor of transforming growth factor alpha (TGF), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGF into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGF. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGF to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.\n\nAbbreviations

Cell Biology↗

bioRxiv2016

Transcriptional pulsing of a nucleolar transgene

The pulsatile nature of transcription has recently emerged as an important property of gene expression. Here we report on the characterization of a RNA polymerase II transgene that is transcribed in the nucleolus. Using the MS2-GFP reporter system and live cell imaging, we found that the synthesis of a MS2-tagged transcript in the nucleolus was discontinuous in all of the cells that were observed, with periods of activity lasting from 15 minutes to 21 hours. The frequency of pulse lengths could be fitted with an exponential function, from which we determined that transcription occurs on average for periods of 20 minutes. These ON periods alternate with periods of inactivity which last on average 29 minutes. The post-mitotic reactivation of transcription was found to be asynchronous in daughter cell pairs. Our observation of discontinuous transcriptional activity in the nucleolus may reflect cycling in the assembly and disassembly of active chromatin structure in and/or around the rDNA genes.

Cell Biology↗

bioRxiv2016

Cdk activity drives senescence from G2 phase

In response to DNA damage a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity is retained after DNA damage until terminal cell cycle exit. The low level of Cdk activity not only allows cell cycle progression, but also forces cell cycle exit at a decision point in G2 phase. We find that Cdk activity stimulates p21 production, leading to nuclear sequestration of Cyclin B1, subsequent APC/CCdh1-dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle drives senescence in the presence of DNA damage, ensuring a robust response when most needed.

Cell Biology↗

bioRxiv2016

Glycolytic enzymes localize to synapses under energy stress to support synaptic function

Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a glycolytic metabolon, that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that energy demands in neurons are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior.

Cell Biology↗

bioRxiv2016

An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. In G2 phase, the arrest is initiated by ATM/ATR-dependent signalling that blocks mitosis-promoting kinases as Plk1. Interestingly, Plk1 can counteract ATR-dependent signalling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of targets including Kap1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing a cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modelling we propose that the minimal duration of a cell cycle arrest is controlled by a timer. Our model shows how cell cycle re-start can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

Cell Biology↗

bioRxiv2016

Engineered kinesin motor proteins amenable to small molecule inhibition

The human genome encodes 45 kinesins that drive cell division, cell motility, intracellular trafficking, and ciliary function. Determining the cellular function of each kinesin would be greatly facilitated by specific small molecule inhibitors, but screens have yielded inhibitors that are specific to only a small number of kinesins, likely due to the high conservation of the kinesin motor domain across the superfamily. Here we present a chemical-genetic approach to engineer kinesin motors that retain microtubule-dependent motility in the absence of inhibitor yet can be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we tested two independent strategies to design inhibitable motors. First, we inserted the six amino acid tetracysteine tag into surface loops of the motor domain such that binding of biarsenic dyes allosterically inhibits processive motility. Second, we fused DmrB dimerization domains to the motor heads such that addition of B/B homodimerizer cross-links the two motor domains and inhibits motor stepping. We show, using cellular assays that the engineered kinesin-1 motors are able to transport artificial and natural kinesin-1 cargoes, but are efficiently inhibited by the addition of the relevant small molecule. Single-molecule imaging in vitro revealed that inhibitor addition reduces the number of processively moving motors on the microtubule, with minor effects on motor run length and velocity. It is likely that these inhibition strategies can be successfully applied to other members of the kinesin superfamily due to the high conservation of the kinesin motor domain. The described engineered motors will be of great utility to dynamically and specifically study kinesin function in cells and animals.

Cell Biology↗

bioRxiv2016

Septins are involved at the early stages of macroautophagy.

Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called as an autophagosome captures longlived proteins, damaged or superfluous organelles and delivers to the lysosome for degradation1. We have identified a novel role for septins in autophagy. Septins are GTP-binding proteins that localize at the bud-neck and are involved in cytokinesis in budding yeast2. We show that septins under autophagy prevalent conditions are majorly localized to the cytoplasm in the form of punctate structures. Further, we report that septins not only localize to pre-autophagosomal structure (PAS) but also to autophagosomes in the form of punctate structures. Interestingly, septins also form small non-canonical rings around PAS during autophagy. Furthermore, we observed that in one of the septin Ts\" mutant, cdc10-5, the anterograde trafficking of Atg9 was affected at the non-permissive temperature (NPT). All these results suggest a role of septins in early stages of autophagy during autophagosome formation.

Cell Biology↗

bioRxiv2016

ALADIN is Required for the Production of Fertile Mouse Oocytes.

Asymmetric cell divisions depend upon the precise placement of the mitotic spindle. In mammalian oocytes, spindles assemble close to the cells centre but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphases. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently, we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygous deficient for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is impaired in a majority of oocytes due to problems in spindle orientation prior to the first meiotic anaphase. Those few oocytes that can mature far enough to be fertilized in vitro are unable to support embryonic development beyond the twocell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Cell Biology↗

bioRxiv2016

Local RhoA Activation Induces Cytokinetic Furrows Independent of Spindle Position and Cell Cycle Stage

Cytokinetic cleavage furrows assemble during anaphase at a site that is dictated by the position of the spindle. The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. While many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. It is not known whether a local zone of RhoA activity is sufficient to induce furrow formation or whether the spindle modulates furrow assembly through other pathways. Similarly, it is not known whether the entire cortex is responsive to RhoA, nor whether contractile ring assembly is cell cycle regulated. Here, we have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation. Light-mediated recruitment of a RhoGEF domain to the plasma membrane leads to rapid activation of RhoA, leading to assembly of cytokinetic furrows that partially ingress. Furthermore, furrow formation in response to RhoA activation is not spatially or temporally restricted. RhoA activation is sufficient to generate furrows at both the cell equator and at cell poles, in both metaphase and anaphase. Remarkably, furrow formation can be initiated in rounded interphase cells, but not adherent cells. These results indicate RhoA activation is sufficient to induce assembly of functional contractile rings and that cell rounding facilitates furrow formation.

Cell Biology↗

bioRxiv2016

Unusual intragenic suppression of an IFT52 gene disruption links hypoxia to the intraflagellar transport in Tetrahymena thermophila.

IFT52 protein is a conserved intraflagellar transport protein (a part of the IFT complex B) that is essential for assembly and maintenance of cilia. Tetrahymena null mutants with an insertion of a neo gene cassette into the IFT52 gene undergo frequent suppressions that lead to conditional assembly of cilia only under hypoxic conditions (Brown et al. 2003). Here we show that these conditional suppressions are intragenic and occur by a novel mechanism. First, the non-native (bacterial) portion of the DNA sequence of the neo cassette is deleted during the process of genome rearrangement that occurs in the developing macronucleus of conjugating Tetrahymena. Next, the residual sequences of the neo cassette (of Tetrahymena origin) within the IFT52 mRNA are recognized as multiple introns and undergo splicing, leading to a restoration of the translational frame of IFT52. The resulting hypoxia-dependent IFT52 protein contains an insertion of 43 new amino acids that replace 7 original amino acids. Taken together with a study in Chlamydomonas reinhardtii showing a hypoxia-dependence of another IFT subunit mutant, IFT46, (Hou et al. 2007), our observations generalize that defective IFT complex subunits can regain functionality under hypoxia.

Cell Biology↗

bioRxiv2016

Effects of Unleaded Petroleum on the Macrophage Aggregates (MA) formation in Red King tilapia (Oreochromis sp.) Fingerlings

The Philippines is one of the major producers of tilapia, the most cultured fish and widely consumed in the world. Although fishes in general is said to be adapted to various stressful conditions, the effect on several cellular immune parameters may be of interest to determine the capacity of the organism to withstand stressors. In this paper, the effect of unleaded petroleum on the splenic macrophage aggregate (MA) formation was studied. This was done to have an overview of the immune response of Tilapia or fishes in general when an oil spill, which almost occur annually at different parts of the world, happen. Histological analysis assessed the area occupied by splenic MA 24 hours after introduction of unleaded petroleum to the aquatic system. To determine whether Mabuhay balls, a technology that claims to be beneficial in terms of improving water quality, was added to one tank (T1) to be able to compare it with another tank (T2). There is a strong statistically significant difference between the groups at day1 (p=0.000) opposite the result of day 6 (p=0.155). Thus, unleaded petroleum increased MA formation, a sign that may indicate a high immune activity as an initial positive response to stress. Mabuhay ball have lessen the mortality but has no effect on splenic MA formation.

Cell Biology↗

bioRxiv2016

An atypical role for the myeloid receptor Mincle in CNS injury

Mincle is a C-type lectin known to play a role in innate immune responses to sterile inflammation, but its contribution to pathologies following an ischemic or traumatic injury is not well understood. In the current study we demonstrate a key role for Mincle in ischemic (i.e. transient middle cerebral artery occlusion) but not traumatic central nervous system injury; absence of Mincle also did not significantly alter the extent of tissue damage or functional outcome in peripheral models of ischemic tissue injury. In the stroke model mice lacking Mincle displayed significantly improved functional outcome from focal cerebral ischemia. The functional improvements in Mincle KO animals were accompanied by reduced infiltration of neutrophils and lower levels of proinflammatory cytokines in recruited peripheral blood cells. Bone marrow chimera experiments revealed that presence of Mincle in the central nervous system, but not peripheral immune cells, was the critical regulator of a poor outcome following transient focal cerebral ischemia, however we exclude a direct role for Mincle in microglia or neural activation. We demonstrate that Mincle lacks widespread expression in the brain, but is specifically associated with macrophages resident in the perivascular niche. These findings implicate Mincle in the initiation, extent and severity of local responses to ischemic injury in the brain, but not peripheral tissues. Mincle signalling therefore offers a novel therapeutic target in the quest to limit damage after stroke.\n\nSources of supportAustralian National Health & Medical Research Council [1057846, 1060538 and Fellowship to NAR], SpinalCure Australia (Career Development Fellowship to MJR), the Australian Research Council, the State Government of Victoria, the Australian Government and The University of Queensland.

Cell Biology↗

bioRxiv2016

Growth Rate-Dependent Global Amplification of Gene Expression

Regulation of cell growth rate is essential for maintaining cellular homeostasis and survival in diverse conditions. Changes in cell growth rate result in changes in rRNA and tRNA content, but the effect of cell growth rate on mRNA abundance is not known. We developed a new method for measuring absolute transcript abundances using RNA-seq, SPike in-based Absolute RNA Quantification (SPARQ), that does not assume a constant transcriptome size and applied it to the model eukaryote, Saccharomyces cerevisiae (budding yeast), grown at different rates. We find that increases in cell growth rate result in increased absolute abundance of almost every transcript, with significant coordinated changes in abundances among functionally related transcripts. mRNA degradation and synthesis rates increase with increased growth rate, but to differing extents, resulting in the observed net increases in absolute abundance. We propose that regulation of ribosome abundance links environmental conditions to transcriptome amplification via nutrient-sensing pathways.

Cell Biology↗