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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 253 records · Page 14Linked to original sources

bioRxiv2016

The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia formation via ubiquitylation of CSPP-L.

Primary cilia are crucial for signal transduction in a variety of pathways, including Hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The Ubiquitin-Proteasome System (UPS) component UBR5 was previously identified as a putative modulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 Ubiquitin ligase that is frequently deregulated in tumours, but its biological role in cancer is largely uncharacterised, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.

cell biology↗

bioRxiv2018

Extracellular matrix regulates morphogenesis and function of ciliated sensory organs in Caenorhabditis elegans

Cilia and extracellular vesicles (EVs) are signaling organelles that play important roles in human health and disease. In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. Hence, the nematode offers an excellent system in which to address central questions regarding the biology of cilia, EVs, and the polycystins. We discovered an unexpected role of the mec-1, mec-5, and mec-9 genes encoding extracellular matrix (ECM) components. We determined that these ECM encoding genes regulate polycystin localization and function, ciliary EV release, cilia length, dendritic morphology, and neuron-glia interactions. Abnormal ECM and fibrosis are observed in ciliopathies such as ADPKD, nephronophthisis, and Bardet-Biedl Syndrome. Our studies reveal multifaceted roles for ECM proteins in the ciliated nervous system of the worm and provide a powerful new in vivo model to study the relationship between ECM, the polycystins, and ciliopathies.

cell biology↗

bioRxiv2018

Lateral plasma membrane compartmentalization links protein function and turnover

Biological membranes organize their proteins and lipids into nano- and microscale patterns. In the yeast plasma membrane (PM) constituents segregate into a large number of distinct domains. However, if and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, biochemical function and endocytic turnover. Using the methionine permease Mup1 as model system we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner Nce102 and TORC2 signaling. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM. Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.

cell biology↗

bioRxiv2017

An experimental and computational framework to build a dynamic protein atlas of human cell division

Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, timing of interactions and changes in cellular structure are all crucial to ensure correct assembly, function and regulation of protein complexes1-4. Live cell imaging can reveal protein distributions and dynamics but experimental and theoretical challenges prevented its use to produce quantitative data and a model of mitosis that comprehensively integrates information and enables analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries.\n\nTo address this, we generated a 4D image data-driven, canonical model of the morphological changes during mitotic progression of human cells. We used this model to integrate dynamic 3D concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here in the context of the MitoSys consortium to generate a dynamic protein atlas of human cell division is generic. It can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types and can be conceptually transferred to other cellular functions.

systems biology↗

bioRxiv2017

Lipid perturbation compromises UPR-mediated ER homeostasis as a result of premature degradation of membrane proteins

BackgroundPhospholipid homeostasis in biological membranes is essential to maintain functions of organelles such as the endoplasmic reticulum. Phospholipid perturbation has been associated to non-alcoholic fatty liver disease, obesity and other metabolic disorders. However, in most cases, the biological significance of lipid disequilibrium remains unclear. Previously, we reported that Saccharomyces cerevisiae adapts to lipid disequilibrium by upregulating several protein quality control pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and the unfolded protein response (UPR).\n\nResultsSurprisingly, we observed certain ER-resident transmembrane proteins (TPs), which form part of the UPR programme, to be destabilised under lipid perturbation (LP). Among these, Sbh1 was prematurely degraded by fatty acid remodelling and membrane stiffening of the ER. Moreover, the protein translocon subunit Sbh1 is targeted for degradation through its transmembrane domain in an unconventional Doa10-dependent manner.\n\nConclusionPremature removal of key ER-resident TPs might be an underlying cause of chronic ER stress in metabolic disorders.

cell biology↗

bioRxiv2018

Active fluctuations modulate gene expression in mouse oocytes

In mammals, the nucleus is central in oocytes, not defining the future embryo axis. Nucleus centring depends on an F-actin mediated pressure gradient. In Fmn2-/- oocytes, lacking the F-actin nucleator Formin 2, the nucleus is off-centre and can be centred by re-expressing Formin 2. Here, we addressed the biological significance of nucleus positioning in mammalian oocytes. Using a dedicated computational 3D imaging approach, we observed nuclear architecture alterations in mouse Fmn2-/- oocytes. RNA sequencing of control versus Fmn2-/- oocytes detected 2285 mis-regulated genes. Rescue experiments showed that the process of nuclear positioning impacts nuclear architecture and gene expression. Using signal processing methods coupled to biophysical modelling allowing the extraction of in vivo mechanical properties of the nuclear envelope, we showed that F-actin-mediated activity promotes nuclear envelope shape fluctuations and chromatin motion. We thus propose a mechano-transduction model whereby nucleus positioning via microfilaments modulates oocyte transcriptome, essential for further embryo development.

cell biology↗

bioRxiv2018

Biotinylation by proximity labelling favours unfolded proteins

Folded enzymes are essential for life, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. Intrinsically Disordered Regions (IDRs) are enriched in disease-linked and multiply post-translationally modified proteins. The extent of foldability of predicted IDRs is difficult to measure due to significant technical challenges to survey in vivo protein conformations on a proteome-wide scale. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than more ordered protein regions, if they retain their flexibility in vivo. Indeed, we observed a positive correlation of predicted IDRs and biotinylation density across four independent large-scale proximity proteomics studies that together report >20 000 biotinylation sites. We show that biotin painting is a promising approach to fill gaps in knowledge between static in vitro protein structures, in silico disorder predictions and in vivo condition-dependent subcellular plasticity using the 80S ribosome as an example.

cell biology↗

bioRxiv2018

Aurora A depletion reveals centrosome-independent polarization mechanism in C. elegans

How living systems break symmetry in an organized manner is an important question in biology. In C. elegans zygotes, symmetry breaking normally occurs in the vicinity of centrosomes, resulting in anterior-directed cortical flows and establishment of a single posterior PAR-2 domain. Here, we report that zygotes depleted of the Aurora A kinase AIR-1 or of centrosomes establish two posterior domains, one at each pole. Using transgenic animals and microfabricated triangular chambers, we establish that such bipolarity occurs in a PAR-2- and curvature-dependent manner. Furthermore, we develop an integrated physical model of symmetry breaking, establishing that local PAR-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for self-organized PAR polarization without functional centrosomes in C. elegans.\n\nOne Sentence SummaryWe uncover a novel centrosome-independent mechanism of polarization in C. elegans zygotes

cell biology↗

bioRxiv2018

Lamin A/C controls nuclear matrin-3 levels and localization, but not alternative splicing of cassette exons

Disruptions in connections between the nuclear lamina and nuclear matrix occur in myopathic disorders. However, the biological significance of nuclear lamina - nuclear matrix coupling still remains largely undetermined. Previously it has been demonstrated that the nuclear matrix protein, matrin-3, binds to lamin A/C and this interaction is disrupted in laminopathies resulting in enhanced separation between the lamina and matrix. Matrin-3 has recently been identified as a core regulator of alternative splicing, whereas the involvement of lamin A/C in splicing still remains controversial. In this study, we demonstrate that lamin A/C is not only required for maintaining the nuclear organization of matrin-3, but also of other splicing activators and small nuclear ribonucleoproteins (snRNP) components. Interestingly, mis-localization of these splicing components did not appear to significantly disrupt alternative splicing events of cassette exons regulated by matrin-3. Thus, the lamin A/C-matrin3 interaction is unlikely to be involved in controlling alternative splicing but could be important in coordinating other nuclear activities. Interestingly, matrin-3 knock-down results in misshapen nuclei suggesting its interaction with lamin A/C maybe important in maintaining nuclear structural integrity.

cell biology↗

bioRxiv2017

HIVs Feedback Circuit Breaks The Fundamental Limit On Noise Suppression To Stabilize Fate

Diverse biological systems utilize gene-expression fluctuations ( noise) to drive lineage-commitment decisions1-5. However, once a commitment is made, noise becomes detrimental to reliable function6,7 and the mechanisms enabling post-commitment noise suppression are unclear. We used time-lapse imaging and mathematical modeling, and found that, after a noise-driven event, human immunodeficiency virus (HIV) strongly attenuated expression noise through a non-transcriptional negative-feedback circuit. Feedback is established by serial generation of RNAs from post-transcriptional splicing, creating a precursor-product relationship where proteins generated from spliced mRNAs auto-deplete their own precursor un-spliced mRNAs. Strikingly, precursor auto-depletion overcomes the theoretical limits on conventional noise suppression--minimizing noise far better than transcriptional auto-repression--and dramatically stabilizes commitment to the active-replication state. This auto-depletion feedback motif may efficiently suppress noise in other systems ranging from detained introns to non-sense mediated decay.

cell biology↗

bioRxiv2018

Homeostatic remodeling of mammalian membranes in response to dietary lipids is essential for cellular fitness

Biological membranes form the functional, dynamic interface that hosts a major fraction of all cellular bioactivity. Proper membrane physiology requires maintenance of a narrow range of physicochemical properties, which must be buffered from external perturbations. While homeostatic adaptation of membrane fluidity to temperature variation is a ubiquitous design feature of ectothermic organisms, such responsive membrane adaptation to external inputs has not been directly observed in mammals. Here, we report that challenging mammalian membrane homeostasis by dietary lipids leads to robust lipidomic remodeling to preserve membrane physical properties. Specifically, exogenous polyunsaturated fatty acids (PUFAs) are rapidly and extensively incorporated into membrane lipids, inducing a reduction in membrane packing. These effects are rapidly compensated both in culture and in vivo by lipidome-wide remodeling, most notably upregulation of saturated lipids and cholesterol. These lipidomic changes result in recovery of membrane packing and permeability. This lipidomic and biophysical compensation is mediated in part by lipid regulatory machinery, whose pharmacological or genetic abrogation results in cytotoxicity when membrane homeostasis is challenged by dietary lipids. These results reveal an essential mammalian mechanism for membrane homeostasis wherein lipidome remodeling in response to dietary lipid inputs preserves functional membrane phenotypes.

cell biology↗

bioRxiv2018

Csmd2 is a Synaptic Transmembrane Protein that Interacts with PSD-95

Mutations and copy number variants of the Cub and Sushi Multiple Domains 2 (CSMD2) gene are associated with schizophrenia and autism spectrum disorder. CSMD2 is a single-pass transmembrane protein with a large extracellular domain comprising repeats of Cub and Sushi domains. Although the biological functions of CSMD2 have not been studied, the association between CSMD2 variants and cognitive function suggest that it may have a role in brain development or function. In this study, we show that mouse Csmd2 is expressed in excitatory and inhibitory neurons in the brain. Csmd2 protein exhibits a somatodendritic localization in the neocortex and hippocampus, with smaller puncta localizing further out in the neuropil. We show that many of these Csmd2 puncta co-localize with the synaptic protein PSD-95. Using immunohistochemical and biochemical methods, we further demonstrate that Csmd2 localizes to dendritic spines and is enriched in the postsynaptic density. We also find Csmd2 at ribbon synapses of the inner plexiform layer of the retina, suggesting a broader synaptic function of Csmd2 in the central nervous system. Finally, we show that the cytoplasmic tail domain of Csmd2 interacts with synaptic scaffolding proteins of the membrane-associated guanylate kinase (MAGUK) family. The association between Csmd2 and MAGUK member PSD-95 is dependent on a PDZ-binding domain on the Csmd2 tail, which is also required for synaptic targeting of Csmd2. Together, these results point toward a function for Csmd2 in dendrites and synapses, which may account for its association with several psychiatric disorders.

cell biology↗

bioRxiv2018

Shorter telomere length is associated with a more recent diagnosis of coeliac disease

BackgroundCoeliac disease (CD) is an autoimmune disease that causes an inappropriate inflammatory immune response to dietary gluten. Telomere length is a marker of biological ageing and is reduced in several autoimmune conditions. This observational study measured salivary telomere length (TL) in gluten-free diet (GFD) treated CD individuals to determine if CD, and length of time on a GFD, is associated with salivary TL.\n\nMethodsClinical and demographic information was collected from CD individuals currently treated with a GFD and healthy non-affected controls. Only participants aged under 35 years at recruitment were included. Relative telomere length was measured using quantitative PCR in oral mucosa collected from saliva. Linear regression was used to determine whether salivary TL was associated with CD, or length of time on a GFD, adjusting for age and sex.\n\nResultsThis study included 79 participants, 52 GFD-treated CD and 27 non-affected controls. No significant difference in salivary TL between individuals with treated CD and controls was found. Within CD individuals, salivary TL was associated with length of time on a GFD, with individuals who started a GFD [≤]3 years ago having shorter salivary TL compared to those who started a GFD > 3 years ago (0.37{+/-}0.05 vs 0.50{+/-}0.04; p=0.002).\n\nConclusionOur findings indicate that salivary TL shorten while CD is untreated, however following treatment on a GFD, they appear to recover to those seen in unaffected controls. This highlights the importance of early diagnosis and initiation of GFD to minimise mucosal damage and telomere shortening, to enable TL to recover.

cell biology↗

bioRxiv2016

Nano Random Forests to mine protein complexes and their relationships in quantitative proteomics data

The large and ever increasing numbers of quantitative proteomics datasets constitute a currently underexploited resource for drawing biological insights on proteins and their functions. Multiple observations by different laboratories indicate that protein complexes often follow similar trends. However, proteomic data is often noisy and incomplete - members of a complex may correlate weakly or only in a fraction of all experiments, or may not be observed in all experiments. We have previously used the Random Forest (RF) machine-learning algorithm to distinguish functional chromosomal proteins from hitchhikers in an analysis of mitotic chromosomes. Even though it is assumed that RFs need large training sets, in this technical note we show that RFs also are able to detect small protein complexes and relationships between them. We use artificial datasets to demonstrate the robustness of RFs to identify small groups even when working with mixes of noisy and apparently uninformative experiments. We then use our procedure to retrieve a number of chromosomal complexes from real quantitative proteomics datasets, comparing wild-type and multiple different knock-out mitotic chromosomes. The procedure also revealed other proteins that covary strongly with these complexes suggesting novel functional links. Integrating the RF analysis for several complexes revealed the known interdependency of kinetochore subcomplexes, as well as an unexpected dependency between the Constitutive-Centromere-Associated Network (CCAN) and the condensin (SMC 2/4) complex. Serving as negative control, ribosomal proteins remained independent of kinetochore complexes. Together, these results show that this complex-oriented RF (nanoRF) can uncover subtle protein relationships and higher-order dependencies in integrated proteomics data.

Cell Biology↗

bioRxiv2013

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

The canonical protein tyrosine phosphatase PTP1B has traditionally been considered to exclusively reside on the endoplasmic reticulum (ER). Using confocal microscopy, we show that endogenous PTP1B actually exhibits a higher local concentration at the mitochondria in all mammalian cell lines that we tested. Fluorescently labeled chimeras containing full-length PTP1B or only its 35 amino acid tail anchor localized identically, demonstrating the complete dependence of PTP1Bs subcellular partitioning on its tail anchor. Correlative light and electron microscopy using GFP-driven photo-oxidation of DAB revealed that PTP1Bs tail anchor localizes it to the mitochondrial interior and to mitochondrial-associated membrane (MAM) sites along the ER. Heterologous expression of the tail anchor of PTP1B in the yeast S. cerevisiae surprisingly led to its exclusive localization to the ER/vacuole with no presence at the mitochondria. Studies with various yeast mutants of conserved membrane insertion pathways revealed a role for the GET/TRC40 pathway in ER insertion, but also emphasized the likely dominant role of spontaneous insertion. Further studies of modified tail isoforms in both yeast and mammalian cells revealed a remarkable sensitivity of subcellular partitioning to slight changes in transmembrane domain (TMD) length, C-terminal charge, and hydropathy. For example, addition of a single positive charge to the tail anchor was sufficient to completely shift the tail anchor to the mitochondria in mammalian cells and to largely shift it there in yeast cells, and a point mutation that increased TMD hydropathy was sufficient to localize the tail anchor exclusively to the ER in mammalian cells. Striking differences in the subcellular partitioning of a given tail anchor isoform in mammalian versus yeast cells most likely point to fundamental differences in the lipid composition of specific organelles (e.g. affecting membrane charge or thickness) in higher versus lower eukaryotes. Fluorescence lifetime imaging microscopy (FLIM) detection of the Forster Resonance Energy Transfer (FRET)-based interaction of the catalytic domain of PTP1B with the epidermal growth factor receptor (EGFR/ErbB1) at the mitochondria revealed a strong interaction on the cytosolic face of the outer mitochondrial membrane (OMM), suggesting the presence of a significant pool of PTP1B there and a novel role for PTP1B in the regulation of mitochondrial ErbB1 activity. In summary, in addition to its well-established general localization along the ER, our results reveal that PTP1B specifically accumulates at MAM sites along the ER and localizes as well to the OMM and mitochondrial matrix. Further elucidation of PTP1Bs roles in these different locations (including the identification of its targets) will likely be critical for understanding its complex regulation of general cellular responses, cell proliferation, and diseased states.

Cell Biology↗

bioRxiv2013

p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery of proteasome activity. We previously demonstrated that the transcription factor Nrf1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER-membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol.

Cell Biology↗

bioRxiv2014

An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. Here we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be \"restarted\" in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown.

Cell Biology↗

bioRxiv2014

OPPOSING MICROTUBULE MOTORS CONTROL MOTILITY, MORPHOLOGY, AND CARGO SEGREGATION DURING ER-TO-GOLGI TRANSPORT.

We recently demonstrated that dynein and kinesin motors drive multiple aspects of endosomal function in mammalian cells. These functions include driving motility, maintaining morphology (notably through providing longitudinal tension to support vesicle fission), and driving cargo sorting. Microtubule motors drive bidirectional motility during traffic between the endoplasmic reticulum (ER) and Golgi. Here, we have examined the role of microtubule motors in transport carrier motility, morphology, and domain organization during ER-to-Golgi transport. We show that consistent with our findings for endosomal dynamics, microtubule motor function during ER-to-Golgi transport of secretory is required for motility, morphology of, and cargo sorting within vesicular tubular carriers en route to the Golgi. Our data are consistent with previous findings that defined roles for dynein-1 and kinesin-1 (KIF5B) and kinesin-2 in this trafficking step. Our high resolution tracking data identify some intriguing aspects. Depletion of kinesin-1 reduces the number of motile structures seen which is in line with other findings relating to the role of kinesin-1 in ER export. However, those transport carriers that were produced had a much greater run length suggesting that this motor can act as a brake on anterograde motility. Kinesin-2 depletion did not significantly reduce the number of motile transport carriers but did cause a similar increase in run length. These data suggest that kinesins act as negative regulators of ER-to-Golgi transport. Depletion of dynein not only reduced the number of motile carriers formed but also caused tubulation of carriers similar to that seen for SNX-coated early endosomes. Our data indicated that the previously observed anterograde-retrograde polarity of transport carriers in transit to the Golgi from the ER is maintained by microtubule motor function.

Cell Biology↗