SEARCH · bioRxiv Science

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources

bioRxiv2017

Sympathetic Transmitters Controlling Thermogenic Efficacy of Brown Adipocytes by Modulating Mitochondrial Complex V

Obesity is epidemic worldwide as the results of excessive energy intake or inefficient energy expenditure. It is promising to utilize the thermogenic function of brown adipose tissue for obesity intervention. However, the mechanisms controlling the efficacy of norepinephrine-induced thermogenesis in brown adipocytes remain elusive. Here, we demonstrate that the proton ATPase activity of mitochondrial complex V is a key factor, which antagonizes proton leak by UCP1 and determines the efficacy of norepinephrine-induced thermogenesis in brown adipocytes. Furthermore, to avoid unnecessary and undesired heat production, we reveal that ATP as a sympathetic cotransmitter is necessary for the high efficacy and specificity of norepinephrine-induced thermogenesis in brown adipocytes by upregulating the ATP synthase activity of complex V. Thus, we demonstrate the modulation mechanism of thermogenic efficacy in brown adipocytes. These findings imply new strategies for efficiently utilizing brown adipocytes thermogenic capacity, and therapeutic targets for the treatments of obesity and diabetes.\n\nHighlights1. Norepinephrine (NE) induces heterogeneous responses in brown adipocytes\n\n2. NE activates the H+-ATPase activity of mitochondrial complex V\n\n3. Mitochondrial complex V is a key factor in NE-induced thermogenic efficacy\n\n4. ATP as a sympathetic cotransmitter enhances the NE-induced thermogenic efficacy

cell biology↗

bioRxiv2017

Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

cell biology↗

bioRxiv2017

CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a high-throughput method for tracking any locus of interest is desirable. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions. Compared to alternatives, our method does not require a nearby repetitive sequence and it requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

cell biology↗

bioRxiv2017

Rapid isolation of functionally intact nuclei from the yeast Saccharomyces

Most available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmatic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.

cell biology↗

bioRxiv2017

cAMP Signaling Regulates DNA Demethylation by Augmenting the Intracellular Labile Ferrous Iron Pool

It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA demethylation.

cell biology↗

bioRxiv2017

Soluble Syntaxin 3 Functions as a Transcription Regulator

Syntaxins - a conserved family of SNARE proteins - contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. Alternative splicing leads to a soluble isoform, termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5, targets to the nucleus and interacts physically and functionally with several transcription factors, including ETV4 and ATF2. Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer vs. normal breast tissue. Inhibition of endogenous Stx3S expression leads to changes in the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.

cell biology↗

bioRxiv2017

Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

The Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking the nanos homologues nos-1 and nos-2 iC. elegans. nos-1nos-2 PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. The nos-1nos-2 phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in a nos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.

cell biology↗

bioRxiv2017

Cell-nonautonomously tunable actomyosin flowsorient distinct cell division axes

Cell division axes during animal development are arranged in diverse orientations, but the molecular mechanisms underlying this diversity remain unclear. By focusing on oriented divisions that are independent of known microtubule/dynein pathways, we show here that the non-muscle myosin II motor is an extrinsically tunable force generator that orients cell division axes through cortical actomyosin flows. We identified three extracellular cues that generate different actomyosin flows. A single contact site locally inhibited myosin activity in a mechanosensitive manner to generate local flow asymmetry, while size asymmetry of two contact sites and Wnt signaling both polarized myosin activity and actomyosin flow, with the latter overriding mechanosensitive effects. These intracellular actomyosin flow anisotropies specify distinct division axes to establish the geometries of not only Caenorhabditis elegans 4-, 6-, and 7-cell stage but also mouse 4-cell stage embryos. Tunable actomyosin flows together with microtubule/dynein pathways may specify diverse division axes across species.

cell biology↗

bioRxiv2017

The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

Why the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.\n\nSummary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

cell biology↗

bioRxiv2017

Rapid DNA Replication Origin Licensing Protects Stem Cell Pluripotency

Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

cell biology↗

bioRxiv2017

Compensation For Chronic Oxidative Stress In ALADIN Null Mice

BackgroundMutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress and several in vitro studies demonstrated that the nucleoporin ALADIN is involved in the cellular oxidative stress response and in adrenal steroidogenesis. We showed that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. Thus, we hypothesized that application of chronic oxidative stress by ingestion of paraquat will generate triple A-like phenotype in ALADIN null mice.\n\nResultsWe demonstrate that ALADIN knock-out mice present with an unexpected compensated glutathione metabolism still lacking a phenotype resembling human triple A syndrome after application of chronic oxidative stress. We could not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN.\n\nConclusionsThis study stresses the species-specific role of the nucleoporin ALADIN presenting a novel compensatory mechanism of the cellular glutathione redox response and shedding light on the role of ALADIN in the cell.

cell biology↗

bioRxiv2017

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a \"local inhibitor\" predicted and described in widely accepted mathematical models, but not previously identified in living cells.

cell biology↗

bioRxiv2017

Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes / lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono - ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral - but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non - ubiquitinatable mutant of Stx3 (Stx3 - 5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV / exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3 - 5R mutant acts as a dominant - negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3 - 5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.

cell biology↗

bioRxiv2017

Rational cell culture optimization enhances experimental reproducibility in cancer cells.

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data.\n\nBaseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce.\n\nWe then devised metabolically rationalized standard assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.

cell biology↗

bioRxiv2017

ESCRT-Independent Surface Receptor And Transporter Protein Degradation By The ILF Pathway

Surface receptor and transporter protein down-regulation drives cell signaling, quality control and metabolism underlying diverse physiology. After endocytosis, proteins are delivered to endosomes where ESCRTs package them into intralumenal vesicles, which are degraded by acid hydrolases upon fusion with lysosomes. However, reports of ESCRT-independent surface protein degradation are emerging suggesting that alternative, non-canonical pathways exist. Using Saccharomyces cerevisiae as a model, here we show that in response to substrates, protein misfolding or TOR signaling, some internalized surface transporters (Hxt3, Itr1, Aqr1) bypass ESCRTs en route to the lysosome membrane where they are sorted into an area that is internalized as an intralumenal fragment (ILF) and degraded upon organelle fusion. This ILF pathway also degrades typical ESCRT client proteins (Mup1, Can1, Ste3) when ESCRT function is impaired. As the underlying machinery is conserved, we speculate that the ILF pathway is an important contributor to receptor and transporter down-regulation in all eukaryotes.

cell biology↗

bioRxiv2017

The mitotic spindle is chiral due to torques generated by motor proteins

Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins1,2. Forces are required for the congression of chromosomes to the metaphase plate and separation of chromatids in anaphase3-6. However, torques may also exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. The average helicity of the bundles with respect to the spindle axis is 1.2{degrees}/m. Inactivation of kinesin-5 (Eg5/Kif11) abolished the chirality of the spindle, suggesting that this motor generates the helical shape of microtubule bundles. To explain the observed shapes, we introduce a theoretical model for the balance of forces and torques acting in the spindle, and show that torque is required to generate the helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its architecture.

cell biology↗

bioRxiv2017

The cytoplasmic DNA sensor cGAS promotes mitotic cell death

The cyclic GMP-AMP (cGAMP) synthase cGAS counteracts infections by detecting and binding foreign cytoplasmic DNA1. DNA-induced synthesis of cGAMP activates innate immune signalling and apoptosis through the cGAMP receptor STING and the downstream effector IRF31-7. During interphase the nuclear envelope protects chromosomal self-DNA from cGAS, but the consequences of exposing chromosomes to cGAS following mitotic nuclear envelope disassembly are unknown. Here we demonstrate that cGAS associates with chromosomes during mitosis and binds nucleosomes with even higher affinity than naked DNA in vitro. Nucleosomes nevertheless competitively inhibit the DNA-dependent stimulation of cGAS, and accordingly, chromosomal cGAS does not affect mitotic progression under normal conditions. This suggests that nucleosomes prevent the inappropriate activation of cGAS during mitosis by acting as a signature of self-DNA. During prolonged mitotic arrest, however, cGAS becomes activated to promote cell death, limiting the fraction of cells that can survive and escape mitotic arrest induced by the chemotherapeutic drug taxol. Induction of mitotic cell death involves cGAMP synthesis by cGAS, as well as signal transduction to IRF3 by STING. We thus propose that cGAS plays a previously unappreciated role in guarding against mitotic errors, promoting cell death during prolonged mitotic arrest. Our data also indicate that the cGAS pathway, whose activity differs widely among cell lines, impacts cell fate determination upon treatment with taxol and other anti-mitotic drugs. Thus, we propose the innate immune system may be harnessed to selectively target cells with mitotic abnormalities.

cell biology↗

bioRxiv2017

AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

AKT is a kinase that regulates numerous cellular processes in the brain and mutations in AKT are known to affect brain function. AKT is indirectly implicated in synaptic plasticity, but its direct role has not been studied. Moreover, three highly related AKT isoforms are expressed in the brain, but their individual roles are poorly understood. We find that each AKT isoform has a unique expression pattern in the hippocampus, with AKT1 and AKT3 primarily in neurons but displaying local differences, while AKT2 is in astrocytes. We also find isoform-specific roles for AKT in multiple paradigms of hippocampal synaptic plasticity. AKT1, but not AKT2 or AKT3, is required for L-LTP through regulating activity-induced protein synthesis. Interestingly, AKT activity inhibits mGluR-LTD, with overlapping functions for AKT1 and AKT3. In summary, our studies identify distinct expression patterns and roles in synaptic plasticity for AKT isoforms in the hippocampus.

cell biology↗