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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 487 records · Page 27Linked to original sources

bioRxiv2017

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena

SUMMARY STATEMENTOur study demonstrates compositional and structural differences of the nuclear pore complex between the functionally differentiated macronucleus and micronucleus within a single cytoplasm of ciliated protozoa.\n\nABSTRACTThe nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs.

cell biology↗

bioRxiv2017

Reconciling Conflicting Models for Global Control of Cell-Cycle Transcription

How the program of periodic cell-cycle transcription is controlled has been debated for several years. Models have ranged from control by a CDK-APC/C oscillator, by a transcription factor (TF) network, or by coupled CDK-APC/C and TF networks. In contrast to current models, a recent study concluded that the cell-cycle transcriptional program is primarily controlled by a CDK-APC/C oscillator with little input from the TF network. This conclusion was largely based on an assumption that substantial drops in transcript levels of network TFs would render them unable to regulate their targets. By combining quantitative modeling and an unbiased analysis of the RNA-seq data, we demonstrate that the data from this recent study are completely consistent with previous reports indicating a critical role of a TF network. Moreover, we report substantial transcript dynamics in cells arrested with intermediate levels of B-cyclins, further supporting the model in which oscillating CDK activity is not required to produce phase-specific transcription.

cell biology↗

bioRxiv2017

Genotoxin-induced transcriptional repression regulates selective protein aggregation

Upon genotoxic stress, dynamic relocalization events control DNA repair, and alterations of the transcriptome and proteome enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen for genome maintenance proteins that move under stress, we find that, upon alkylation stress, the splicing factor Hsh155 localizes to both intranuclear and cytoplasmic protein quality control aggregates. Under stress, an ordered sequestration of Hsh155 occurs at nuclear and then cytoplasmic aggregates in a manner that is regulated by molecular chaperones. This dynamic behavior is preceded by a decrease in splicing efficiency. While DNA replication stress signaling is not required for Hsh155 sequestration, Hsh155 aggregation is cell cycle and TOR pathway dependent. Indeed, loss of a TORC1 regulated ribosomal protein gene transcription factor Sfp1 allows general aggregate formation but prevents Hsh155 recruitment. Together, our analyses suggest a model in which some proteins evicted from chromatin undergoing transcriptional remodeling during stress are targeted to protein quality control sites.

cell biology↗

bioRxiv2017

Single-cell RNA-Sequencing uncovers transcriptional states and fate decisionsin haematopoiesis

The success of marker-based approaches for dissecting haematopoiesis in mouse and human is reliant on the presence of well-defined cell-surface markers specific for diverse progenitor populations. An inherent problem with this approach is that the presence of specific cell surface markers does not directly reflect the transcriptional state of a cell. Here we used a marker-free approach to computationally reconstruct the blood lineage tree in zebrafish and order cells along their differentiation trajectory, based on their global transcriptional differences. Within the population of transcriptionally similar stem and progenitor cells our analysis revealed considerable cell-to-cell differences in their probability to transition to another, committed state. Once fate decision was executed, the suppression of transcription of ribosomal genes and up-regulation of lineage specific factors coordinately controlled lineage differentiation. Evolutionary analysis further demonstrated that this haematopoietic program was highly conserved between zebrafish and higher vertebrates.

cell biology↗

bioRxiv2017

Trabecular Meshwork Failure In A Model Of Pigmentary Glaucoma

Pigment dispersion syndrome can lead to pigmentary glaucoma (PG), a poorly understood condition of younger, myopic eyes with fluctuating, high intraocular pressure (IOP). The absence of a model similar in size and behavior to human eyes has made it difficult to investigate its pathogenesis. Here, we present a porcine ex vivo model that recreates the features of PG including intraocular hypertension, pigment accumulation in the trabecular meshwork and relative failure of phagocytosis. In in vitro monolayer cultures as well as in ex vivo eye perfusion cultures, we found that the trabecular meshwork (TM) cells that regulate outflow, form actin stress fibers and have a decreased phagocytosis. Gene expression microarray and pathway analysis indicated key roles of RhoA in regulating the TM cytoskeleton, motility, and phagocytosis thereby providing new targets for PG therapy.

cell biology↗

bioRxiv2017

Semisynthetic pH-Sensitive Fluorophores For Imaging Exocytosis And Endocytosis

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe semisynthetic pH-sensitive protein conjugates that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

cell biology↗

bioRxiv2017

Cytosolic ribosomes on the surface of mitochondria

By electron cryo-tomography and subtomogram averaging, translation-arrested ribosomes were used to depict the clustered organisation of the TOM complex on the surface of mitochondria, corroborating earlier reports of localized translation. Ribosomes were shown to interact specifically with the TOM complex and nascent chain binding was crucial for ribosome recruitment and stabilization. Ribosomes were bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport, and the importance of spatial organization for efficient mitochondrial protein import.

cell biology↗

bioRxiv2017

Conserved roles of RECQ-like helicases Sgs1 and BLM in preventing R-loop induced genome instability

Sgs1 is a yeast DNA helicase functioning in DNA replication and repair, and is the orthologue of the human Blooms syndrome helicase BLM. Here we analyze the mutation signature associated with SGS1 deletion in yeast, and find frequent copy number changes flanked by regions of repetitive sequence and high R-loop forming potential. We show that loss of SGS1 increases R-loop accumulation and sensitizes cells to replication-transcription collisions. Accordingly, in sgs1{Delta} cells the genome-wide distribution of R-loops shifts to known sites of Sgs1 action, replication pausing regions, and to long genes. Depletion of the orthologous BLM helicase from human cancer cells also increases R-loop levels, and R-loop-associated genome instability. In support of a direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop mediated genome instability.

cell biology↗

bioRxiv2017

Can Human Embryonic Stem Cell-Derived Stromal Cells Serve A Starting Material For Myoblasts?

A large number of myocytes is necessary to treat intractable muscular disorders such as Duchenne muscular dystrophy with cell-based therapies. However, starting materials for cellular therapy products such as myoblasts, marrow stromal cells, menstrual blood-derived cells and placenta-derived cells have a limited lifespan and cease to proliferate in vitro. From the viewpoints of manufacturing and quality control, cells with a long lifespan are more suitable as a starting material. In this study, we generated stromal cells for future myoblast therapy from a working cell bank of human embryonic stem cells (ESCs). The ESC-derived CD105+ cells with extensive in vitro proliferation capability exhibited myogenesis and genetic stability in vitro. These results imply that ESC-derived CD105+ cells are another cell source for myoblasts in cell-based therapy for patients with genetic muscular disorders. Since ESCs are immortal, mesenchymal stromal cells generated from ESCs can be manufactured at a large scale in one lot for pharmaceutical purposes.

cell biology↗

bioRxiv2017

Local Nucleation Of Microtubule Bundles Through Tubulin Concentration Into A Condensed Tau Phase

Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that under conditions of macro-molecular crowding, tau forms liquid drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles enclosed in a liquid sheath of tau. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers, and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.

cell biology↗

bioRxiv2017

Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.\n\nOne-sentence summaryThe p38 MAP kinase pathway coordinates cell growth and cell cycle progression by lengthening G1 in small cells, allowing them more time to grow before their next division.

cell biology↗

bioRxiv2017

The phosphatase PTPRG controls FGFR1 activity and influences sensitivity to FGFR kinase inhibitors

FGFR1 represents an important target for precision medicine and a detailed molecular understanding of the target is important in order to increase the efficacy of FGFR inhibitors. We have here applied proximity labelling of FGFR1 in an osteosarcoma cell line to identify determinants of FGFR1 activity. Many known FGFR interactors were identified (e.g. FRS2, PLC{gamma}, RSK2, SHC4, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.

cell biology↗

bioRxiv2017

Mitochondrial replacement in an iPSC model of Leber Hereditary Optic Neuropathy.

Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

cell biology↗

bioRxiv2017

H+- and Na+- elicited swift changes of the microtubule system in the biflagellated green alga Chlamydomonas

The microtubule cytoskeletal system is integral to diverse cellular processes. Although microtubules are known for dynamic instability, the system is tightly controlled in typical interphase animal cells. In contrast, diverse evidence suggests that the system is mercurial in the unicellular fresh water green alga, Chlamydomonas, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by fluctuation of intracellular H+ and Na+. These results suggest disparate sensitivity of this vital yet delicate system in diverse organisms; and illuminate how pH may drive crucial cellular processes; how plants respond to, and perhaps sense stresses; and how many species could be susceptible to accelerated changes in global environments.

cell biology↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗

bioRxiv2017

Localised Dynactin Protects Growing Microtubules To Deliver Oskar mRNA To The Posterior Cortex Of The Drosophila Oocyte

The localisation of oskar mRNA to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Kinesin 1 transports oskar mRNA to the oocyte posterior along a polarised microtubule cytoskeleton that grows from non-centrosomal microtubule organising centres (ncMTOCs) along the anterior/lateral cortex. Here we show that the formation of this polarised microtubule network also requires the posterior regulation of microtubule growth. A mutation in the Dynactin Arp1 subunit causes most oskar mRNA to localise in the posterior cytoplasm rather than cortically. oskar mRNA transport and anchoring are normal in this mutant, but the microtubules fail to reach the posterior pole. Thus, Dynactin acts as an anti-catastrophe factor that extends microtubule growth posteriorly. Kinesin 1 transports Dynactin to the oocyte posterior, creating a positive feedback loop that increases the length and persistence of the posterior microtubules that deliver oskar mRNA to the cortex.

cell biology↗

bioRxiv2017

Drosophila Short stop as a paradigm for the role and regulation of spectraplakins

Spectraplakins are evolutionarily well conserved cytoskeletal linker molecules that are true members of three protein families: plakins, spectrins and Gas2-like proteins. Spectraplakin genes encode at least 7 characteristic functional domains which are combined in a modular fashion into multiple isoforms, and which are responsible for an enormous breadth of cellular functions. These functions are related to the regulation of actin, microtubules, intermediate filaments, intracellular organelles, cell adhesions and signalling processes during the development and maintenance of a wide variety of tissues. To gain a deeper understanding of this enormous functional diversity, invertebrate genetic model organisms, such as the fruit fly Drosophila, can be used to develop concepts and mechanistic paradigms that can inform the investigation in higher animals or humans. Here we provide a comprehensive overview of our current knowledge of the Drosophila spectraplakin Short stop (Shot). We describe its functional domains and isoforms and compare them with those of the mammalian spectraplakins dystonin and MACF1. We then summarise its roles during the development and maintenance of the nervous system, epithelia, oocytes and muscles, taking care to compare and contrast mechanistic insights across these functions in the fly, but especially also with related functions of dystonin and MACF1 in mostly mammalian contexts. We hope that this review will improve the wider appreciation of how work on Drosophila Shot can be used as an efficient strategy to promote the fundamental concepts and mechanisms that underpin spectraplakin functions, with important implications for biomedical research into human disease.

cell biology↗

bioRxiv2017

A Di-Acidic Motif Targets Cytoplasmic Proteins For Unconventional Protein Secretion

We previously reported that Acb1, a cytoplasmic protein in Saccharomyces cerevisiae that cannot enter the endoplasmic reticulum (ER), was secreted upon nutrient starvation by a Vps4 independent, but ESCRT-I, -II and -III and Grh1 dependent pathway (Curwin et al., 2016). Here, we report that the same conditions result in secretion of another signal sequence lacking protein, superoxide dismutase 1 (SOD1). Similar to Acb1, SOD1 export requires Grh1 and a subset of ESCRT components. Importantly, our analysis reveals the existence of a conserved di-acidic motif (Asp-Glu) in SOD1 and Acb1 that is required for their respective export. This sequence is different from the di-acidic motif (Asp-X-Glu) necessary for export of transmembrane proteins from the ER. We propose that the Asp-Glu sequence acts as a targeting motif for the entry of SOD1 and Acb1, and likely many other proteins, upon nutrient starvation into a common albeit ER-Golgi independent pathway of secretion.

cell biology↗