SEARCH · Search bioRxiv

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 919 records · Page 51Linked to original sources

bioRxiv2017

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near the centrosome, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

cell biology↗

bioRxiv2018

Dynamin-2 facilitates Atg9 recycling from nascent autophagosomes

Autophagy involves rapid growth of phagophores through membrane addition. Newly added membranes are derived from other organelles through vesicles carrying the Atg9 protein. Membrane is delivered by fusing these vesicles with the phagophores. Atg9 is, nevertheless, not incorporated in autophagosomes. We now show that this protein is retrieved from phagophores by fission utilizing Dynamin-2 (Dnm2) as the membrane scission protein. Blocking Atg9 recycling by interfering with Dnm2 helps retain Atg9 in autophagosomes and degrades this protein by autophagy. Dnm2 colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1) when autophagy is induced, consistent with transient interactions during Atg9 retrieval. EndoB1 and Dnm2 also control the downstream fusion of phagophores to late endosomes, thus ensuring the completion of phagophores before proceeding to the next stage in the autophagy process. These data provide novel insights into the roles of membrane scission proteins during autophagy.

cell biology↗

bioRxiv2018

Phospholipid flipping facilitates annexin translocation across membranes

Annexins are phospholipid binding proteins that somehow translocate from the inner leaflet of the plasma membrane to the outer leaflet. For example, Annexin A2 is known to localise to the outer leaflet of the plasma membrane (cell surface) where it is involved in plasminogen activation leading to fibrinolysis and cell migration, among other functions. Despite having well described extracellular functions, the mechanism of annexin transport from the cytoplasmic inner leaflet to the extracellular outer leaflet of the plasma membrane remains unclear. Here, we show that phospholipid flipping activity is crucial for the transport of annexins A2 and A5 across membranes in cells and in liposomes. We identified TMEM16F (anoctamin-6) as a lipid scramblase required for transport of these annexins to the outer leaflet of the plasma membrane. This work reveals a mechanism for annexin translocation across membranes which depends on plasma membrane phospholipid flipping.

cell biology↗

bioRxiv2018

Gemin4 is an essential gene in mice, and its overexpression in human cells causes relocalization of the SMN complex to the nucleoplasm

Gemin4 is a member of the Survival Motor Neuron (SMN) protein complex, which is responsible for the assembly and maturation of Sm-class small nuclear ribonucleoproteins (snRNPs). In metazoa, Sm snRNPs are assembled in the cytoplasm and subsequently imported into the nucleus. We previously showed that the SMN complex is required for snRNP import in vitro, although it remains unclear which specific components direct this process. Here, we report that Gemin4 overexpression drives SMN and the other Gemin proteins from the cytoplasm into the nucleus. Moreover, it disrupts the subnuclear localization of the Cajal body marker protein, coilin, in a dose-dependent manner. We identified three putative nuclear localization signal (NLS) motifs within Gemin4, one of which is necessary and sufficient to direct nuclear import. Overexpression of Gemin4 constructs lacking this NLS sequestered Gemin3 and, to a lesser extent Gemin2, in the cytoplasm but had little effect on the nuclear accumulation of SMN. We also investigated the effects of Gemin4 depletion in the laboratory mouse, mus musculus. Gemin4 null mice die early in embryonic development, demonstrating that Gemin4 is an essential mammalian protein. When crossed onto a severe SMA mutant background, heterozygous loss of Gemin4 failed to modify the early postnatal mortality phenotype of SMA type I (Smn-/-;SMN2+/+) mice. We conclude that Gemin4 plays an essential role in mammalian snRNP biogenesis, and may facilitate import of the SMN complex (or subunits thereof) into the nucleus.

cell biology↗

bioRxiv2018

CHC22 Clathrin Diverts GLUT4 from the ER-to-Golgi Intermediate Compartment for Intracellular Sequestration

Glucose Transporter 4 (GLUT4) is sequestered inside muscle and fat, then released by vesicle traffic to the cell surface in response to post-prandial insulin for blood glucose clearance. Here we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively-secreted GLUT1 transporter and localize CHC22 to the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4 and sortilin and down-regulation of either p115 or CHC22, but not GM130 or sortilin abrogate insulin-responsive GLUT4 release. This indicates CHC22 traffic initiates human GLUT4 sequestration from the ERGIC, and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.\n\nSummaryBlood glucose clearance relies on insulin-mediated exocytosis of glucose transporter 4 (GLUT4) from sites of intracellular sequestration. We show that in humans, CHC22 clathrin mediates membrane traffic from the ER-to-Golgi Intermediate Compartment, which is needed for GLUT4 sequestration during GLUT4 pathway biogenesis.

cell biology↗

bioRxiv2018

PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation

PARP1 regulates the repair of DNA single strand breaks (SSBs) generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whilst PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication associated DNA damage due to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and through stabilising replication forks that encounter BER intermediates.

cell biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗

bioRxiv2018

Coupling of Rho family GTPases during mesenchymal-to-epithelial-like transitions

Many metazoan developmental processes require cells to transition between migratory mesenchymal- and adherent epithelial-like states. These transitions require Rho GTPase-mediated actin rearrangements downstream of integrin and cadherin pathways. A regulatory toolbox of GEF and GAP proteins precisely coordinates Rho protein activities, yet defining the involvement of specific regulators within a cellular context remains a challenge due to overlapping and coupled activities. Here we demonstrate that Drosophila dorsal closure is a simple, powerful model for Rho GTPase regulation during leading edge to cadherin contact transitions. During these transitions a Rac GEF elmo-dock complex regulates both lamellipodia and Rho1-dependent, actomyosin-mediated tension at initial cadherin contacts. Moreover, the Drosophila Rho GAP arhgap21 ortholog controls Rac and Rho GTPases during the same processes and genetically regulates the elmo-dock complex. This study presents a fresh framework to understand the inter-relationship between GEF and GAP proteins that tether Rac and Rho cycles during developmental processes.

cell biology↗

bioRxiv2018

Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue

BackgroundMelanoma can be diagnosed by identifying nests of cells on the skin surface. Understanding the processes that drive nest formation is important as these processes could be potential targets for new cancer drugs. Cell proliferation and cell migration are two potential mechanisms that could conceivably drive melanoma nest formation. However, it is unclear which one of these two putative mechanisms plays a dominant role in driving nest formation.\n\nResultsWe use a suite of three-dimensional (3D) experiments in human skin tissue and a parallel series of 3D individual-based simulations to explore whether cell migration or cell proliferation plays a dominant role in nest formation. In the experiments we measure nest formation in populations of irradiated (non-proliferative) and non-irradiated (proliferative) melanoma cells, cultured together with primary keratinocyte and fibroblast cells on a 3D experimental human skin model. Results show that nest size depends on initial cell number and is driven primarily by cell proliferation rather than cell migration.\n\nConclusionsWe find that nest size depends on initial cell number, and is driven primarily by cell proliferation rather than cell migration. All experimental results are consistent with simulation data from a 3D individual based model (IBM) of cell migration and cell proliferation.

cell biology↗

bioRxiv2018

Transcription factor dynamics reveals a circadian code for fat cell differentiation

Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern during stress and disease correlates with increased fat mass and obesity in humans, raising the intriguing question of how hormone secretion dynamics affect the process of adipocyte differentiation. By using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system, leading to very low adipocyte differentiation rates. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushings disease, and other conditions for which glucocorticoid secretion loses its pulsatility can lead to obesity. Given the ubiquitous nature of oscillating hormone secretion in mammals, the filtering mechanism we uncovered may represent a general temporal control principle for differentiation.\n\nHIGHLIGHTO_LIWe found that the fraction of differentiated cells is controlled by rhythmic and pulsatile hormone stimulus patterns.\nC_LIO_LITwelve hours is the cutoff point for daily hormone pulse durations below which cells fail to differentiate, arguing for a circadian code for hormone-induced cell differentiation.\nC_LIO_LIIn addition to fast positive feedback such as between PPARG and CEBPA, the adipogenic transcriptional architecture requires added parallel slow positive feedback to mediate temporal filtering of circadian oscillatory inputs\nC_LI

cell biology↗

bioRxiv2018

The abnormal, mis-localizated HR bmh protein associates with members of the protein processing machinery in the cytoplasm

We have recently mapped a 296 bp deletion at the mouse hairless locus that causes the mutation hairless rhino bald Mill Hill, (Hrrhbmh), (ID:MGI:3039558; J:89321), removing the stop codon and generating a larger mutant protein HR bmh with an additional sequence of 117 amino acids. The mutant hairless gene mRNA is expressed during the embryonic and post-natal development of the hair follicle. A mutant HR protein was identified in bmh mouse skin at different stages of development by a specific antibody. We demonstrated that the HR bmh protein is able to interact with the vitamin D receptor, but is not able to repress VDR-mediated transactivation. Immunofluorescence analysis revealed that HR bmh protein displays an abnormal cellular localization in transfected cell lines, as well as in the epidermis and hair follicle of bmh mutant mice. Here we analyse the patterns of HR bmh extra-nuclear localization in cell transfection experiments. Using a candidate approach, double transfection experiments, immunofluorescent staining and IP protocols we established that HR bmh co-localizes specifically with the proteins HDAC6 in the cytoplasm is able to physically interact with it. Blast analysis allowed to show that HDAC6 and share high sequence homologies with specific motifs of HR bmh. We studied the association of these potential interactors with various cytoplasmic compartments. We discuss the relevance of the mutant hairless protein mis-localization in endosomal processing and in proteasome related pathways with respect to the specific skin phenotype of mouse hairless mutants.

cell biology↗

bioRxiv2018

A conserved function for pericentromeric satellite DNA

A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus. However, the underlying mechanism to ensure such a configuration is unknown. Here we provide evidence that pericentromeric satellite DNA, which is often regarded as junk, is a critical constituent of the chromosome, allowing the packaging of all chromosomes into a single nucleus. We show that the multi AT-hook satellite DNA binding proteins, D. melanogaster D1 and mouse HMGA1, play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into chromocenters, a cytological association of pericentromeric heterochromatin. Defective chromocenter formation leads to micronuclei formation due to budding off of interphase nucleus and cell death. We propose that chromocenter and satellite DNA serves a fundamental role to achieve the universal characteristic of eukaryotic cells: full complement of the genome within a single nucleus.

cell biology↗

bioRxiv2018

CENP-B dynamics at centromeres is regulated by a SUMOylation/ubiquitination and proteasomal-dependent degradation mechanism involving the SUMO-targeted ubiquitin E3 ligase RNF4

Centromeric protein B (CENP-B) is a major constituent of the centromere. It is a DNA binding protein that recognizes a specific 17-nt sequence present in the centromeric alphoid satellite repeats. CENP-B importance for centromere stability has only been revealed recently. In addition to its DNA binding properties, CENP-B interacts with the histone H3 variant CENP-A and CENP-C. These interactions confer a mechanical strength to the kinetochore that enables accurate sister chromatids segregation to avoid aneuploidy. Therefore, understanding the mechanisms that regulate CENP-B stability at the centromere is a major unresolved issue for the comprehension of centromere function. In this study, we demonstrate that lysine K402 of CENP-B is a substrate for SUMO post-translational modifications. We show that K402 regulates CENP-B stability at centromeres through a SUMOylation/ubiquitination and proteasomal-dependent degradation mechanism involving the SUMO-Targeted Ubiquitin E3 Ligase RNF4/SNURF. Our study describes SUMOylation of CENP-B as a major post-translational modification involved in centromere dynamics.

cell biology↗

bioRxiv2018

Local Protein Dynamics During Microvesicle Exocytosis in Neuroendocrine Cells

Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is locally recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

cell biology↗

bioRxiv2018

Detection of Pro-apoptotic BaxΔ2 Proteins in the Human Cerebellum

Bax{Delta}2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax{Delta}2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax{Delta}2 expression in human tissues, we examined a panel of human brain samples. Here, we report 4 cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax{Delta}2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax{Delta}2- positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax{Delta}2 is consistent with that known for Baxa, but in a less ubiquitous manner, these results may imply a potential function of Bax{Delta}2 in Purkinje cells.

cell biology↗

bioRxiv2018

Src-transformed cells hijack mitosis to extrude from the epithelium

At the initial stage of carcinogenesis single mutated cells appear within an epithelium. Mammalian in vitro experiments show that potentially cancerous cells undergo live apical extrusion from normal monolayers. However, the mechanism underlying this process in vivo remains poorly understood. Mosaic expression of the oncogene vSrc in a simple epithelium of the early zebrafish embryo results in apical extrusion of transformed cells. Here we find that during extrusion components of the cytokinetic ring are recruited to adherens junctions of transformed cells, stimulating formation of a misoriented pseudo cytokinetic ring. During extrusion, the ring constricts and separates the basal from the apical part of the cell releasing both from the epithelium. This process requires cell cycle progression and occurs immediately after vSrc-transformed cell enters mitosis. To achieve extrusion, vSrc coordinates cell cycle progression, junctional integrity, cell survival and apicobasal polarity. Without vSrc, modulating these cellular processes reconstitutes vSrc-like extrusion, confirming their sufficiency for this process.

cell biology↗

bioRxiv2018

Cell transformation disrupts the efficiency of chromosome segregation through microtubule detyrosination

The general principles of chromosome segregation are highly conserved throughout eukaryotic evolution. However, it is unknown whether there are differences in spindle or kinetochore composition or architecture which influence the efficiency chromosome segregation in different cell types. Here we show that the transition of human retinal pigment epithelial cells to a mesenchymal phenotype causes a stabilisation of kinetochore-microtubule attachments and an increase in the frequency of chromosome mis-segregation, due to inefficient error-correction, during mitosis. We find that this is caused by microtubule detyrosination during the epithelial-to-mesenchymal transition and that parthenolide, a tubulin carboxypeptidase inhibitor, efficiently reverts mes-enchymal cells to the epithelial mode of chromosome segregation. We propose that reprogramming the post-translational modifications of the mitotic spindle decreases mitotic fidelity and may contribute to CIN in mesenchymal cell populations during tumorigenesis.

cell biology↗

bioRxiv2018

The effects of mechanical strain on mouse eye lens capsule and cellular microstructure

The understanding of multiscale load transfer within complex soft tissues is incomplete. The eye lens is ideal for multiscale tissue mechanics studies as its principal function is to fine focus light at different distances onto the retina via mechanical shape changes. The biomechanical function, resiliency, and intricate microstructure of the lens make it an excellent non-connective soft tissue model. We hypothesized that compressive strain applied onto whole lens tissue leads to deformation of specific microstructures and that this deformation is reversible following removal of load. For this examination, mouse lenses were compressed by sequential application of increasing load. Using confocal microscopy and quantitative image analysis, we determined that axial strain [≥]10% reduces capsule thickness, expands epithelial cell area, and separates fiber cell tips at the anterior region of the lenses. At the equatorial region, strain [≥]6% increases fiber cell widths. The effects of strain on lens epithelial cell area, capsule thickness, and equatorial fiber cell widths are reversible following the release of lenses from strain. However, although fiber cell tip separation following the removal of low loads is reversible, the separation becomes irreversible with application of higher loads. This irreversible separation between fiber cell tips leads to incomplete bulk lens resiliency. The lens is an accessible biomechanical model system that provides new insights on multiscale transfer of loads in soft tissues.

cell biology↗