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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,297 records · Page 72Linked to original sources

bioRxiv2018

Is Actin Filament Sliding Responsible for Endoplasmic Reticulum and Golgi Movement?

The plant secretory pathway is responsible for the production of the majority of proteins and carbohydrates consumed on the planet. The early secretory pathway is composed of Golgi bodies and the endoplasmic reticulum (ER) and is highly mobile in plants with rapid remodelling of the ER network. The dynamics of the ER and Golgi bodies is driven by the actin cytoskeleton and myosin motor proteins play a key role in this. However, exactly how myosin motor proteins drive remodelling in plants is currently a contentious issue. Here, using a combination of live cell microscopy and over-expression of non-functional myosins we demonstrate that myosin motor proteins drive actin filament sliding and subsequently the dynamics of the secretory pathway.\n\nSummaryIn plants, the actin cytoskeleton and myosins are fundamental for normal dynamics of the endomembrane system and cytoplasmic streaming. We demonstrate that this is in part due to myosin driven sliding of actin filaments within a bundle. This generates, at least in part, the motive force required for cell dynamics in planta.

cell biology↗

bioRxiv2018

Glucose increases the lifespan of post-reproductive C. elegans independently of FOXO

Aging is one of the most critical risk factors for the development of metabolic syndromes1. Prominent metabolic diseases, namely type 2 diabetes and insulin resistance, have a strong association with endoplasmic reticulum (ER) stress2. Upon ER stress, the unfolded protein response (UPR) is activated to limit cellular damage by adapting to stress conditions and restoring ER homeostasis3,4. However, adaptive genes upregulated from the UPR tend to decrease with age5. Although stress resistance correlates with increased longevity in a variety of model organisms, the links between the UPR, ER stress resistance, and longevity remain poorly understood. Here, we show that supplementing bacteria diet with 2% glucose (high glucose diet, HGD) in post-reproductive 7-day-old (7DO) C. elegans significantly extend their lifespan in contrast to shortening the lifespan of reproductive 3-day-old (3DO) animals. The insulin-IGF receptor DAF-2 and its immediate downstream target, phosphoinositide 3-kinase (PI3K) AGE-1, were found to be critical factors in extending the lifespan of 7DO worms on HGD. The downstream transcription factor forkhead box O (FOXO) DAF-16 did not extend the lifespan of 7DO worms on HGD in contrast of its previously reported role in modulating lifespan of 3DO worms6. Furthermore, we identified that UPR activation through the highly conserved ATF-6 and PEK-1 sensors significantly extended the longevity of 7DO worms on HGD but not through the IRE-1 sensor. Our results demonstrate that HGD extends lifespan of post-reproductive worms in a UPR-dependent manner but independently of FOXO. Based on these observations, we hypothesise that HGD activates the otherwise quiescent UPR in aged worms to overcome age-related stress and to restore ER homeostasis. In contrast, young adult animals subjected to HGD leads to unresolved ER stress, conversely leading to a deleterious stress response.

cell biology↗

bioRxiv2018

Pivoting of microtubules driven by minus end directed motors leads to their alignment to form an interpolar bundle

At the beginning of mitosis, the cell forms a spindle made of microtubules and associated proteins to segregate chromosomes. An important part of spindle architecture is a set of antiparallel microtubule bundles connecting the two spindle poles. A key question is how microtubules extending at arbitrary angles form an antiparallel interpolar bundle. Here we show that microtubules meet at an oblique angle and subsequently rotate into antiparallel alignment. By combining experiments with theory, we show that microtubules from each pole search for those from the opposite pole by performing random angular movement. Upon contact of two microtubules, they slide sideways along each other towards the minus end, which we interpret as the action of minus end directed Cut7/kinesin-5 motors. In conclusion, random rotational motion helps microtubules from the opposite poles to find each other and subsequent accumulation of motors allows them to generate forces that drive interpolar bundle formation.

cell biology↗

bioRxiv2018

Nuclear speckle fusion via long-range directional motion regulates the number and size of speckles

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significant increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 um and with velocities between 0.2-1.5 m/min. Frequently, 3 or even 4 speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer but larger speckles after transcriptional inhibition. These speckle movements are not actin-dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle-marker protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization.

cell biology↗

bioRxiv2018

Protection of circadian rhythms by the protein folding chaperone, BiP

ER stress and dysregulation of collagen synthesis are associated with progression of disease in cancer and fibrosis. Collagen synthesis is co-ordinated with the circadian clock, which curiously in cancer cells, is deregulated by ER stress. We hypothesised that interplay exists between circadian rhythm, collagen synthesis and ER stress in normal cells. Here we show that fibroblasts with ER stress do not demonstrate circadian rhythms in gene expression upon clock-synchronizing time cues. Conversely, overexpression of BiP or treatment with chemical chaperones strengthens the oscillation amplitude of circadian rhythms. The significance of these findings was explored in tendon, where we showed that BiP expression is ramped preemptively prior to a surge in collagen synthesis at night, thereby preventing protein misfolding and ER stress. In turn, we propose, this forestalls activation of the unfolded protein response in order for circadian rhythms to be maintained. Thus, targeting ER stress could be used to modulate circadian rhythm and restore collagen homeostasis in disease.

cell biology↗

bioRxiv2018

Regulation of the Unfolded Protein Response by BiP AMPylation protects photoreceptors from light-dependent degeneration

In response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hours of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hours once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

cell biology↗

bioRxiv2018

The N-cadherin interactome in primary cardiomyocytes as defined by quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors are comprised primarily of adaptor and adhesion proteins that promote junction specialization. Finally, we find evidence of dynamic interplay between AJ and Z-disc proteins. Together, our results provide novel insight into the cardiomyocyte AJ and provide a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion.\n\nSummary StatementProximity proteomics reveals a specific and specialized N-cadherin (CDH2) interactome along the cell-cell contacts of primary cardiomyocytes.

cell biology↗

bioRxiv2018

14-3-3 recruits keratin intermediate filaments to mechanically sensitive cell-cell contacts

AbstractIntermediate filament cytoskeletal networks simultaneously support mechanical integrity and influence signal transduction pathways. Marked remodeling of the keratin intermediate filament network accompanies collective cellular morphogenetic movements that occur during early embryonic development in the frog Xenopus laevis. While this reorganization of keratin is initiated by force transduction on cell-cell contacts mediated by C-cadherin, the mechanism by which keratin filament reorganization occurs remains poorly understood. In this work we demonstrate that 14-3-3 proteins regulate keratin reorganization dynamics in embryonic mesendoderm cells from Xenopus gastrula. 14-3-3 co-localizes with keratin filaments near cell-cell junctions in migrating mesendoderm. Co-immunoprecipitation, mass spectrometry and bioinformatic analyses indicate Keratin 19 is a target of 14-3-3 in the whole embryo and, more specifically, mesendoderm tissue. Inhibition of 14-3-3 results in both the decreased exchange of keratin subunits into filaments and blocks keratin filament recruitment toward cell-cell contacts. Synthetically coupling 14-3-3 to Keratin 19 through a unique fusion construct conversely induces the localization of this keratin population to the region of cell-cell contacts. Taken together, these findings indicate that 14-3-3 acts on keratin intermediate filaments and is involved in their reorganization to sites of cell adhesion.

cell biology↗

bioRxiv2018

Feedback Regulation between Initiation and Maturation Networks Orchestrates the Chromatin Dynamics of Epidermal Lineage Commitment

Tissue development results from lineage-specific transcription factors (TF) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we interrogate the epigenomic landscape during epidermal differentiation and create an inference network that ranks the coordinate effects of TF-accessible regulatory element-target gene expression triplets on lineage commitment. We discover two critical transition periods: surface ectoderm initiation and keratinocyte maturation, and identify TFAP2C and p63 as lineage initiation and maturation factors, respectively. Surprisingly, we find that TFAP2C, and not p63, is sufficient to initiate surface ectoderm differentiation, with TFAP2C-initiated progenitor cells capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively auto-regulate its expression and close a subset of the TFAP2C-initiated early program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

cell biology↗

bioRxiv2018

Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome and controls the abundance of key regulators of cellular function by selective protein degradation, but how foldingdefective proteins in the secretory system are selected from the large and diverse constellation of membrane and secretory proteins and efficiently delivered to proteasomes in the cytosol is not well understood. To determine the basis of substrate selectivity in human cells, we developed a transcriptional shut off approach to conduct parallel, unbiased, genome-wide CRISPR analysis of structurally and topologically diverse ER-associated degradation (ERAD) clients. Highly quantitative screen metrics allowed precise dissection of entire pathways, enabling identification of unique substrate-specific combinations of recognition and ubiquitin conjugation modules. Our analysis identified cytosolic ubiquitin conjugating machinery that has not been previously linked to ERAD but collaborates with membrane-integrated ubiquitin ligases to conjugate branched or mixed ubiquitin chains to promote efficient and processive substrate degradation.

cell biology↗

bioRxiv2018

Parameter inference with analytical propagators for stochastic models of autoregulated gene expression

Stochastic gene expression in regulatory networks is conventionally modelled via the Chemical Master Equation (CME). As explicit solutions to the CME, in the form of so-called propagators, are oftentimes not readily available, various approximations have been proposed. A recently developed analytical method is based on a separation of scales that assumes significant differences in the lifetimes of mRNA and protein in the network, allowing for the efficient approximation of propagators from asymptotic expansions for the corresponding generating functions. Here, we showcase the applicability of that method to simulated data from a telegraph model for gene expression that is extended with an autoregulatory mechanism. We demonstrate that the resulting approximate propagators can be applied successfully for Bayesian parameter inference in the non-regulated model; moreover, we show that, in the extended autoregulated model, autoactivation or autorepression may be refuted under certain assumptions on the model parameters. Our results indicate that the method showcased here may allow for successful parameter inference and model identification from longitudinal single cell data.

cell biology↗

bioRxiv2018

Gene editing can generate fragile bivalents in mouse oocytes

Mouse female meiotic spindles assemble from acentriolar MTOCs (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOCs biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master MTOC regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

cell biology↗

bioRxiv2018

High glucose-induced ubiquitylation of G6PD leads to the injury of podocyte

Oxidative stress contributes substantially to podocyte injury in diabetic kidney disease. The mechanism of hyperglycemia-induced oxidative stress in podocytes is not fully understood. Glucose-6-phosphate dehydrogenase is critical in maintaining NADPH, an important cofactor for antioxidant system. Here, we hypothesized that high glucose induces ubiquitylation and degradation of G6PD, which injures podocytes by reactive oxygen species (ROS) accumulation. We found that both G6PD protein expression and G6PD activity was decreased in kidneys of both diabetic patients and diabetic rodents. Overexpressing G6PD reversed redox imbalance and podocyte apoptosis induced by high glucose and palmitate. Inhibition of G6PD induced podocyte apoptosis. In G6PD deficient mice, podocyte apoptosis was also largely increased. High glucose had no effect on G6PD mRNA level but it caused decreased G6PD protein expression, which was mediated by the ubiquitin proteasome pathway. Furthermore, von Hippel-Lindau (VHL), an E3 ubiquitin ligase subunit, directly bound to G6PD and degraded G6PD through ubiquitylating G6PD on lysine residues 366/403. Our data suggest that high glucose induces ubiquitylation of G6PD by VHL, which leads to ROS accumulation and podocyte injury.

cell biology↗

bioRxiv2018

17-(Allylamino)-17demethoxygeldanamycin reduces Endoplasmic Reticulum (ER) stress-induced mitochondrial dysfunction in C2C12 myotubes

In patients with myositis, persistent skeletal muscle weakness in the absence of significant inflammatory cell infiltrates is a well-recognised, but poorly understood, cause of morbidity. This has led researchers to investigate cellular mechanisms independent of immune cells, which may contribute to this underlying muscle weakness. Chronic ER stress pathway activation is evident in the muscle of myositis patients, and is now a potential mediator of muscle weakness in the absence of inflammation. Abnormal ER stress pathway activation is associated with mitochondrial dysfunction, resulting in bioenergetic deficits and reactive oxygen species (ROS) generation, which in this context may potentially damage muscle proteins and thus impair contractile performance. This study examined whether treatment with the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17AAG) could mitigate these ER stress-induced changes. C2C12 myotubes were treated with the ER stress-inducing compound Tunicamycin, in the presence or absence of 17AAG. Myotubes were examined for changes relating to ER stress pathway activation, mitochondrial function, markers of oxidative damage and in myotubular dimensions. ER stress pathway activation caused mitochondrial dysfunction, as evidenced by reduced oxygen consumption and ATP generation and by increased gene expression levels of the bio-energetic regulator, uncoupling protein 3 (UCP-3), the latter indicative of electron transport chain uncoupling. ER stress pathway activation also caused increased gene expression of superoxide dismutase (SOD) 2 and peroxiredoxin (PRDX) 3, elevated H2O2 levels, and reduced total thiol pool levels and a significant diminution of myotubular dimensions. Exposure to 17AAG ameliorated these ER stress-induced changes. These findings, which suggest that 17AAG can reduce ER stress-induced mitochondrial dysfunction, oxidative damage and myotubular atrophy, have potential implications in the context of human myositis.

cell biology↗

bioRxiv2018

An Organometallic Ir(III) Molecular Probe for Imaging Microtubules in Fluorescence and Electron Microscopy

We report a versatile cyclometalated Iridium (III) complex probe that achieves synchronous fluorescence-electron microscopy correlation to reveal microtubule ultrastructure in cells. The selective insertion of probe between repeated and {beta} units of microtubule triggers remarkable fluorescent enhancement, and high TEM contrast due to the presence of heavy Ir ions. The highly photostable probe allows live cell imaging of tubulin localization and motion during cell division with an resolution of 20 nm, and under TEM imaging reveals the {beta} unit interspace of 45[A] of microtubule in cells.

cell biology↗

bioRxiv2018

Kif17 phosphorylation regulates its ciliary localization and photoreceptor outer segment turnover

BackgroundKIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown.\n\nResultsUsing transgenic expression in both mammalian cells and zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization to cilia. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding.\n\nConclusionsTaken together, our data support a model in which phosphorylation of KIF17 promotes its ciliary localization. In cone photoreceptor outer segments, this promotes disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

cell biology↗

bioRxiv2018

Three-dimensional organization of transzonal projections and other cytoplasmic extensions in mouse ovarian follicles

Each mammalian oocyte is nurtured by its own multi-cellular structure, the ovarian follicle. We used new methods for serial section electron microscopy to examine entire cells and their projections in mouse antral ovarian follicles. It is already known that cumulus cells send towards the oocyte thin cytoplasmic projections called transzonal projections (TZPs), which are crucial for normal oocyte development. We found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the connected TZPs are usually contacted on their shaft by oocyte microvilli. Mural granulosa cells were found to possess randomly oriented cytoplasmic projections that are strikingly similar to free-ended TZPs. We propose that granulosa cells use cytoplasmic projections to search for the oocyte, and cumulus cell differentiation results from a contact-mediated paracrine interaction with the oocyte.

cell biology↗

bioRxiv2018

Endoplasmic reticulum stress may activate NLRP3 inflammasomes via TXNIP in preeclampsia

Preeclampsia (PE) development is often associated with placental immune and inflammatory dysregulation, as well as endoplasmic reticulum (ER) stress. However, the mechanisms linking ER stress and inflammatory dysregulation to PE have not been clarified. It has been reported that thioredoxin-interacting protein (TXNIP), which can bind with and activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome, plays a critical role in immune regulation. Recent experimental evidence suggests that activated NLRP3 inflammasomes can activate interleukin-1{beta} (IL-1{beta}) production in the placenta of patients with PE. The objective of the current study was to explore if TXNIP plays a critical signaling role linking ER stress with NLRP3 inflammasome activation in PE. We hypothesised that ER stress would induce TXNIP production, which would bind with NLRP3 inflammasomes to activate IL-1{beta} production. HTR8/SVneo cells were subjected to six hours hypoxia followed by six hours reoxygenation (H/R). These cells showed a higher protein level of NLRP3 and IL-1{beta}, as well as a higher enzymatic activity of caspase-1, indicating enhanced inflammatory dysregulation and ER stress. Cells transfected with TXNIP siRNA showed reduced NLRP3 inflammasome activation. Cells treated with 4-phenylbutyric acid, an inhibitor of ER stress, showed a similar result. In addition, the outgrowth of explant with TXNIP lentivirus in H/R or Tunicamycin (inducers of ER stress) was also measured to verify our hypothesis. These findings demonstrated that TXNIP could influence inflammatory dysregulation by mediating ER stress and NLRP3 inflammasome activation in PE. This novel mechanism may further explain the inflammation observed at the maternal-fetal interface, which leads to placental dysfunction in a patient with PE.

cell biology↗