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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 631 records · Page 35Linked to original sources

bioRxiv2017

A transgenic toolkit for visualizing and perturbing microtubules reveals unexpected functions in the epidermis

Differentiation induces reorganization of microtubules (MTs) into non-centrosomal arrays in a variety of tissues. The physiological functions of these microtubule arrays are just beginning to be understood as few tools currently exist to genetically perturb microtubule organization in vivo, particularly in mammals. We developed a genetic toolkit that can be broadly applied to the study of microtubule dynamics and function in many cell types. Using a TRE-EB1-GFP mouse we demonstrate that distinct differentiation transitions in the epidermis cause a decrease in microtubule growth rates and microtubule growth lifetimes, resulting in strong suppression of dynamics. To understand the physiological functions of these stable, non-centrosomal microtubules, we generated a TRE-spastin mouse, which can be used to perturb microtubule organization in a wide-variety of tissues in vivo. Unexpectedly, microtubule perturbation exclusively in post-mitotic keratinocytes had profound consequences on epidermal morphogenesis. We uncoupled novel cell-autonomous roles for MTs in differentiation-driven cell flattening from non-cell autonomous functions in regulating proliferation, differentiation, and tissue architecture. Taken together, we have created tools that will be broadly useful for the study of microtubule dynamics and function in mammalian tissue physiology and have used them to uncover previously unknown functions for non-centrosomal microtubules during mammalian epidermal development.

cell biology↗

bioRxiv2017

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 m thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues.

cell biology↗

bioRxiv2017

Widespread translational remodeling during human neuronal differentiation

Faithful cellular differentiation requires precise coordination of changes in gene expression. However, the relative contributions of transcriptional and translational regulation during human cellular differentiation are unclear. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and characterized genomewide RNA and translation levels during neurogenesis. We find that thousands of genes change at the translation level across differentiation without a corresponding change in RNA level. Specifically, we identify mTOR complex 1 signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by transcript 3' UTRs, through regulatory sequences. Together, our findings identify a functional role for the dramatic 3' UTR extensions that occur during brain development, and provide insights to interpret genetic variants in post-transcriptional control factors that influence neurodevelopmental disorders and diseases.

cell biology↗

bioRxiv2017

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

Genetically encoded Forster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Forster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mChery and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

cell biology↗

bioRxiv2017

Tension-dependent stretching and folding of ZO-1 controls the localization of its interactors

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays we show that the tight junction protein ZO-1 undergoes actomyosin tension-dependent stretching and folding in vivo. Magnetic tweezers experiments using purified ZO-1 indicate that pN-scale tensions (~2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact. Actomyosin tension and substrate stiffness regulate the localization and expression of the transcription factor DbpA and the tight junction membrane protein occludin in a ZO-1/ZO-2-dependent manner, resulting in modulation of gene expression, cell proliferation, barrier function and cyst morphogenesis. Interactions between the N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, and folding is antagonized by heterodimerization with ZO-2. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to modulate their protein interactions and downstream signaling.

cell biology↗

bioRxiv2017

HID-1 controls cargo sorting and dense core formation by influencing trans- Golgi network acidification in neuroendocrine cells

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN) where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.

cell biology↗

bioRxiv2017

Dynamics of the IFT Machinery at the Ciliary Tip

Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using Photogate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

cell biology↗

bioRxiv2017

Caspase-8, RIPK1, and RIPK3 Coordinately Regulate Retinoic Acid-Induced Cell Differentiation and Necroptosis

Caspase-8, which is essential for death receptor-mediated apoptosis, inhibits necroptosis by suppressing the function of RIPK1 and RIPK3 to activate MLKL. We show that knockdown of caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripkl and Ripk3. RA treatment obviously enhanced the expression of RA-specific genes having a retinoic acid response element (RARE) to induce cell differentiation, and induced marked expression of RIPK1, RIPK3 and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RA receptor (RAR) to form a complex with RIPK1 and RIPK3 in the nucleus, and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3. In Caspase-8-deficient mouse embryos, expression of RA-specific genes was obviously enhanced. Thus, caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo.

cell biology↗

bioRxiv2017

Transit and integration of extracellular mitochondria in human heart cells

Tissue ischemia adversely affects the function of mitochondria, which results in impairment of oxidative phosphorylation and compromised recovery of the affected organ. The impact of ischemia on mitochondrial function has been most extensively studied in the heart because of the morbidity and mortality associated with injury to this organ. Because conventional methods to preserve cell viability and function following an ischemic injury are limited in their efficacy, we developed a unique approach to protect the heart by transplanting respiration-competent mitochondria isolated from a non-ischemic tissue to the ischemic region. Our experiments in animals have shown that transplantation of isolated mitochondria to injured heart tissue leads to decreases in cell death, increases in energy production, and improvements in contractile function. We also discovered that exogenously-derived mitochondria injected or perfused into ischemic hearts were readily internalized by cardiac cells through actin-dependent endocytosis. Here, we describe the use of three-dimensional super-resolution microscopy and transmission electron microscopy to determine the intracellular fate of exogenous mitochondria in non-dividing human iPS-derived cardiomyocytes and dividing primary human cardiac fibroblasts. We show isolated mitochondria are internalised in human cardiac cells within minutes and then transported to endosomes and lysosomes. The majority of exogenous mitochondria escape from these compartments and fuse with the endogenous mitochondrial network, while some organelles are degraded through hydrolysis. Understanding this process may guide the development of treatments directed at replacing or augmenting impaired mitochondria in ischemic tissues and provide new options to rejuvenate dysfunctional mitochondria in a wide range of human diseases and disorders.

cell biology↗

bioRxiv2017

Sequential interactions with Mre11-Rad50-Nbs1 activate ATM/Tel1 at DNA double-strand breaks and telomeres

The Mre11-Rad50-Nbs1 (MRN) protein complex, CtIP/Ctp1/Sae2 and ATM/Tel1 kinase protect genome integrity through their functions in DNA double-strand break (DSB) repair, checkpoint signaling, and telomere maintenance. Nbs1 has a conserved C-terminal motif that binds ATM, but the full extent of ATM interactions with MRN are unknown. Here, we show that Tel1 overexpression in Schizosaccharomyces pombe restores Tel1 activity at DSBs and telomeres in the absence of Nbs1. This activity requires Mre11, indicating that Tel1 overexpression drives low affinity binding to the Mre11-Rad50 subcomplex. Mre11-Rad50 binds DSBs in nbs1{Delta} cells, and fusing the Tel1-binding motif of Nbs1 to Mre11 fully restores Tel1 signaling in these cells. Tel1 overexpression does not restore Tel1 signaling in cells carrying the rad50-I1192W mutation, which impairs the ability of Mre11-Rad50 to form the ATP-bound closed conformation. From these findings, we propose that Tel1 activation at DNA ends proceeds by a sequential mechanism initiated by high affinity binding to Nbs1 which recruits Tel1, followed by a low affinity interaction with Mre11-Rad50 in the closed conformation to activate Tel1.

cell biology↗

bioRxiv2017

Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in polynucleotide kinase/phosphatase mutant of fission yeast

The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5-phosphate and 3-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1{Delta}) cells of Schizosaccharomyces pombe. Here, we report that pnk1{Delta} cells have enhanced requirements for Rad3 (ATR/Mec1) and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A ({gamma}H2A) at damaged replication forks. The viability of pnk1{Delta} cells depends on Mre11 and Ctp1 (CtIP/Sae2) double-strand break (DSB) resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1) DNA helicase. Eliminating Pnk1 strongly sensitizes mre11{Delta} pku80{Delta} cells to DNA damaging agents that collapse replication forks, indicating a requirement for Mre11-Rad50-Nbs1 (MRN) protein complex that cannot be efficiently replaced by Exo1 5-3 exonuclease. Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1{Delta} cells, these findings indicate that lingering SSBs in pnk1{Delta} cells trigger Rad51-independent homology-directed repair of collapsed replication forks.\n\nAUTHOR SUMMARYDNA is constantly damaged by normal cellular metabolism, for example production of reactive oxygen species, or from exposure to external DNA damaging sources, such as radiation from the sun or chemicals in the environment. These genotoxic agents create thousands of single-strand breaks/cell/day in the human body. An essential DNA repair protein known as polynucleotide kinase/phosphatase (PNKP) makes sure the single-strand breaks have 5 phosphate and 3 hydroxyl ends suitable for healing by DNA ligase. Mutations that reduce PNKP activity cause a devastating neurological disease but surprisingly not cancer, suggesting that other DNA repair mechanisms step into the breach in dividing PNKP-deficient cells. One popular candidate was homology-directed repair (HDR) of replication forks that collapse at single-strand breaks, but the crucial HDR protein Rad51 was found to be non-essential in PNKP-deficient cells of fission yeast. In this study, Sanchez and Russell revive the HDR model by showing that SSBs in PNKP-deficient cells are repaired by a variant HDR mechanism that bypasses the requirement for Rad51. Notably, Mus81 endonuclease that resolves sister chromatid recombination structures formed during HDR of collapsed replication forks was found to be essential in PNKP-deficient cells.

cell biology↗

bioRxiv2017

Differential activation of Fyn kinase distinguishes saturated and unsaturated fats in mouse macrophages

Diet-induced obesity is associated with increased adipose tissue activated macrophage numbers. Yet, how macrophages integrate fatty acid (FA) signals remains unclear. We previously demonstrated that Fyn deficiency (FynKO) protects against high fat diet-induced adipose tissue macrophage accumulation. Herein, we show that inflammatory markers and reactive oxygen species are not induced in FynKO bone marrow-derived macrophages exposed to the saturated FA palmitate, suggesting that Fyn regulates macrophage function in response to FA signals. Saturated palmitate activates Fyn and re-localizes Fyn into the nucleus of RAW264.7, J774 and wild-type bone marrow-derived macrophages. Similarly, Fyn activity is increased in cells of AT stromal vascular fraction of high fat-fed control mice, with Fyn protein being located in the nucleus of these cells. We demonstrate that Fyn modulates palmitate-dependent oxidative stress in macrophages. Moreover, Fyn catalytic activity is necessary for its nuclear re-localization and downstream effects, as Fyn pharmacological inhibition abolishes palmitate-induced Fyn nuclear redistribution and palmitate-dependent increase of oxidative stress markers. Importantly, mono-or polyunsaturated FAs do not activate Fyn, and fail to re-localize Fyn to the nucleus. Together these data demonstrate that macrophages integrate nutritional FA signals via a differential activation of Fyn that distinguishes, at least partly, the effects of saturated versus unsaturated fats.

cell biology↗

bioRxiv2017

A Cautionary Tail: Changes in Integrin Behavior with Labeling

Genetic expression of fluorescently labeled proteins is essential to visualizing dynamic behavior within live cells. Recent advances in microscopy have increased resolution to the level where it is now possible to capture individual molecules interacting. However, the criteria for determining whether a fluorescent label perturbs protein function have not undergone a corresponding increase in resolution. The effects of protein labeling on cell function are still judged by whether populations of protein localize and interact with known binding partners. Here we use integrins, bidirectional signal adhesion molecules that regulate interactions between the extracellular matrix and the cytoskeleton through a well-defined series of conformational changes to show that not all labeling strategies are the same. We found that labeling the beta subunit decreased the mobility of individual integrin molecules and the protrusive activity of the entire cell. While integrins with labeled alpha subunits behaved similarly to unlabeled integrins, labeling the beta subunit increased the size of adhesions by elevating integrin affinity and exposing the ligand induced binding domain to change the molecule conformation. Thus, our single molecule and cellular data indicate that the ability of labeled proteins to localize and interact with known binding partners does not guarantee it does not alter protein function. We propose that the behaviors of individual molecules rather than the ensemble behavior of populations need to be considered as criteria to determine if a probe is non-perturbative.

cell biology↗

bioRxiv2017

Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling

Toll-like receptor 4 interactor with leucine-rich repeats (Tril) is a transmembrane protein that functions as a coreceptor for Toll-like receptors (Tlrs) to mediate innate immune responses in the adult brain. Tril also triggers degradation of the Bmp inhibitor, Smad7, during early embryonic development to allow for normal blood formation. Tril most likely plays additional, yet to be discovered, roles during embryogenesis. In the current studies, we performed a structure-function analysis, which indicated that the extracellular domain, including the fibronectin type III (FN) domain, and the intracellular domain of Tril are required to trigger Smad7 degradation in the early Xenopus embryo. Furthermore, we found that a Tril deletion mutant lacking the FN domain (Tril{Delta}FN) can dominantly inhibit signaling by endogenous Tril when overexpressed in vivo. This finding raises the intriguing possibility that the FN domain functions to bind endogenous Tril/Tlr4 ligands, perhaps including extracellular matrix molecules. We also show that Tril normally cycles between the cell surface and endosomes, and that the Tril extracellular domain is required to retain Tril at the cell surface, while the intracellular domain is required for Tril internalization in Xenopus ectodermal explants. Using a CHO cell aggregation assay, we further show that, unlike other transmembrane proteins that contain leucine rich repeats in the extracellular domain, Tril is not sufficient to mediate homophilic adhesion. Our findings identify Tril{Delta}FN as a valuable tool that can be used to block the function of endogenous Tril in vivo in order to discover additional roles during embryonic development.

cell biology↗

bioRxiv2017

C3G dynamically associates with Nuclear speckles and regulates mRNA splicing

C3G (RapGEF1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleo-cytoplasmic exchange. Here we show that C3G localizes to SC35 positive nuclear speckles, and regulates splicing activity. Reversible association of C3G with speckles was seen upon inhibition of transcription and splicing. C3G shows partial colocalization with SC35, and is recruited to a chromatin and RNase sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton, and is lost upon expression of the kinase, Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles and inactivation of Rap signalling by expression of GFP- Rap1GAP, alters speckle morphology and number. Enhanced association of C3G with speckles is seen upon GSK3{beta} inhibition, or differentiation of C2C12 cells to myotubes. CRISPR/Cas9 mediated knockdown of C3G resulted in decreased splicing activity and reduced staining for SC35 in speckles. C3G knockout clones of C2C12 as well as MDA-MB- 231 showed reduced protein levels of several splicing factors compared to control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.\n\nSummaryNuclear speckles are sites for pre-mRNA splicing. We provide evidence for localization and function of a Ras family GTPase, Rap1 and its exchange factor C3G in nuclear speckles.

cell biology↗

bioRxiv2017

Biomechanical control of lysosomal secretion via the VAMP7 hub: a tug-of-1 war mechanism between VARP and LRRK1

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we found that cells deficient in the secretory lysosome v-SNARE VAMP7 were impaired in adapting to substrate rigidity. Conversely VAMP7-mediated secretion was stimulated by more rigid substrate and this regulation depended on the Longin domain of VAMP7. We further found that the Longin domain bound the kinase and retrograde trafficking adaptor LRRK1 and LRRK1 negatively regulated VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controlled the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. We propose a mechanism whereby biomechanical constraints regulate VAMP7- dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral readily- releasable pool of secretory lysosomes.

cell biology↗

bioRxiv2017

Context-dependent deposition and regulation of mRNAs in P-bodies

Cells respond to stress by remodeling their transcriptome through transcription and degradation. Xrn1p-dependent degradation in P-bodies is the most prevalent pathway. Yet, P-bodies may facilitate not only decay but also act as storage compartment. However, which and how mRNAs are selected into different degradation pathways and what determines the fate of any given mRNA in P-bodies remain largely unknown. We devised a new method to identify both common and stress-specific mRNA subsets associated with P-bodies. mRNAs targeted for degradation to P-bodies, decayed with different kinetics. Moreover, the localization of a specific set of mRNAs to P-bodies under glucose deprivation was obligatory to prevent decay. Depending on its client mRNA, the RNA binding protein Puf5p either promoted or inhibited decay. The Puf5p-dependent storage of a subset of mRNAs in P-bodies under glucose starvation may be beneficial with respect to chronological lifespan.

cell biology↗

bioRxiv2017

Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/160374v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@67f3aaorg.highwire.dtl.DTLVardef@ae2235org.highwire.dtl.DTLVardef@47f729org.highwire.dtl.DTLVardef@b3c9d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗