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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

HiPLA: High-throughput imaging Proximity Ligation Assay

Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the antibody-based proximity ligation assay in a high-throughput imaging screening format to systematically probe protein interactomes. Using HiPLA, we probe the interaction of 60 proteins and associated PTMs with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. In combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions were accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

cell biology↗

bioRxiv2018

Single molecule analysis of lamin dynamics

The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins (NETs) and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin A {Delta}50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate how pathogenic mutations or cellular processes affect protein dynamics at the NE.

cell biology↗

bioRxiv2018

The palmitoyl acyltransferase DHHC5 mediates beta-adrenergic signaling in the heart by targeting Gα proteins and G protein-coupled receptor kinase 2

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here we investigated whether {beta}-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that {beta}-adrenergic stimulation led to increased Gs and Gi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and positive inotropic responses. Additionally, we identified for the first time that G protein-coupled receptor kinase 2 (GRK2) is a palmitoylated protein in cardiomyocytes. The kinetics of GRK2 palmitoylation were distinct from those observed with Gs and Gi after {beta}-adrenergic stimulation. Knockdown of the plasma membrane-localized palmitoyl acyltransferase DHHC5 revealed that this enzyme is necessary for palmitoylation of Gs, Gi, and GRK2 and functional responses downstream of {beta}-adrenergic stimulation. Our results reveal that DHHC5 activity is required for signaling downstream of {beta}-adrenergic receptors.

cell biology↗

bioRxiv2018

The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid binding domain, recognition of the 3-splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1, showing that it binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysomal profiling indicates the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8 which contributes to metastasis, inflammation, and cancer progression in mice and humans.

cell biology↗

bioRxiv2018

Sulforaphane Alters the Acidification of the Vacuole to Trigger Cell Death

Sulforaphane (SFN) is a compound [1-isothiocyanato-4-(methylsulfinyl)- butane] found in broccoli and other cruciferous vegetables that is currently of interest because of its potential as a chemopreventive and a chemotherapeutic drug. Recent studies in a diverse range of cellular and animal models have shown that SFN is involved in multiple intracellular signaling pathways that regulate cell death, cell cycle progression, and cell invasion. In order to better understand the mechanisms of action behind SFN-induced cell death, we undertook an unbiased genome wide screen with the yeast knockout (YKO) library to identify SFN sensitive (SFNS) mutants. Our mutants were enriched with knockouts in genes linked to vacuolar function suggesting a link between this organelle and SFNs mechanism of action in yeast. Our subsequent work revealed that SFN increases the vacuolar pH of yeast cells and that varying the vacuolar pH can alter the sensitivity of yeast cells to the drug. In fact, several mutations that lower the vacuolar pH in yeast actually made the cells resistant to SFN (SFNR). Finally, we show that human lung cancer cells with more acidic compartments are also SFNR suggesting that SFNs mechanism of action identified in yeast may carry over to higher eukaryotic cells.

cell biology↗

bioRxiv2018

A novel generic dictionary-based denoising method for improving noisy and densely packed nuclei segmentation in 3D time-lapse fluorescence microscopy images

Time-lapse fluorescence microscopy is an essential technique for quantifying various characteristics of cellular processes,i.e. cell survival, migration, and differentiation. To perform high-throughput quantification of cellular processes, nuclei segmentation and tracking should be performed in an automated manner. Nevertheless, nuclei segmentation and tracking are challenging tasks due to embedded noise, intensity inhomogeneity, shape variation as well as a weak boundary of nuclei. Although several nuclei segmentation approaches have been reported in the literature, dealing with embedded noise remains the most challenging part of any segmentation algorithm. We propose a novel denoising algorithms, based on sparse coding, that can both enhance very faint and noisy nuclei but simultaneously detect nuclei position accurately. Furthermore our method is based on a limited number of parameters,with only one being critical, which is the approximate size of the objects of interest. We also show that our denoising method coupled with classical segmentation method works properly in the context of the most challenging cases. To evaluate the performance of the proposed method, we tested our method on two datasets from the cell tracking challenge. Across all datasets, the proposed method achieved satisfactory results with 96.96% recall for C.elegans dataset. Besides, in Drosophila dataset, our method achieved very high recall (99.3%).

cell biology↗

bioRxiv2018

Phosphorylation of Arp2 is not essential for Arp2/3 complex activity in fission yeast

LeClaire et al. presented evidence that phosphorylation of three sites on the Arp2 subunit activates Arp2/3 complex to nucleate actin filaments. We mutated the homologous residues of Arp2 (Y198, T233 and T234) in the fission yeast genome to amino acids that preclude or mimic phosphorylation. Arp2/3 complex is essential for the viability of fission yeast, yet strains unable to phosphorylate these sites grew normally. Y198F/T233A/T234A Arp2 was only nonfunctional if GFP-tagged, as observed by LeClaire et al. in Drosophila cells. Replacing both T233 and T234 with aspartic acid was lethal, suggesting that phosphorylation might be inhibitory. Nevertheless, blocking phosphorylation at these sites had the same effect as mimicking it: slowing assembly of endocytic actin patches. Mass spectrometry revealed phosphorylation at a fourth conserved Arp2 residue, Y218, but both blocking and mimicking phosphorylation of Y218 only slowed actin patch assembly slightly. Therefore, phosphorylation of Y198, T233, T234 and Y218 is not required for the activity of fission yeast Arp2/3 complex.\n\nSummaryPrevious research concluded that phosphorylation at three sites on Arp2 is necessary to activate Arp2/3 complex. Epstein et al. make genomic substitutions blocking or mimicking phosphorylation to demonstrate that phosphorylation of these three sites does not regulate Arp2/3 complex in fission yeast.

cell biology↗

bioRxiv2018

Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III) driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae. In a mutant compromised in RNAP III activity there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.

cell biology↗

bioRxiv2018

Plasticity in the glucagon interactome reveals novel proteins that regulate glucagon secretion in alpha TC1-6 cells

Glucagon is stored within secretory granules of pancreatic alpha cells until a stimulus, such as a change in microenvironmental conditions, triggers its release. We hypothesized that the secretory response of the alpha cell to various stimuli could be determined by plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. To answer this question, we isolated secretory granules from alpha TC1-6 cells and identified glucagon-interacting proteins by affinity purification coupled with liquid chromatography/tandem mass spectrometry. Proteomic analyses revealed a network of cytoplasmic and histone proteins. Specifically, the interaction between glucagon and histone H4 and the ER stress protein GRP78 was confirmed through co-immunoprecipitation of secretory granule lysates, and co-localization within secretory granules using high-resolution confocal microscopy. The composition of these networks was altered at different glucose levels (25 mM vs 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. Finally, siRNA-mediated silencing of a subset of nonhistone proteins revealed novel proteins that may regulate glucagon secretion. We have therefore described a novel and dynamic glucagon interactome within alpha cell secretory granules, and suggest that plasticity in the interactome governs the alpha cell secretory response to paracrine and nutritional stimuli.

cell biology↗

bioRxiv2018

An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1

Fatty acyl-CoA reductase 1 (Far1) is an ubiquitously expressed peroxisomal membrane protein generating fatty alcohols required for the biosynthesis of ether lipids.\n\nLipid droplet localization of human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization and N-glycosylation tagging suggest that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either pex19 or TRC40/ASNA1 CRISPR/Cas9 knockout cells.\n\nIn conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

cell biology↗

bioRxiv2018

Mother centrioles are dispensable for deuterosome formation and function during basal body amplification

Mammalian epithelial cells use a pair of mother centrioles (MCs) and numerous deuterosomes as platforms for efficient basal body assembly during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to either arise spontaneously followed by maturation into larger ones with increased procentriole-producing capacity or be assembled solely on the young MC, nucleate procentrioles under the MCs guidance, and released as procentriole-occupied \"halos\". Here we show that both MCs are dispensable for deuterosome formation in multiciliate cells. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm were initially procentriole-free and then grew into halos. More importantly, eliminating the young MC or both MCs in proliferating precursor cells through shRNA-mediated depletion of Plk4, a kinase essential to procentriole assembly, did not abolish deuterosome formation when these cells were induced to differentiate into mEPCs. The average deuterosome numbers per cell only reduced by 21% as compared to control mEPCs. Therefore, MC is not essential to the assembly of both deuterosomes and deuterosome-mediated procentrioles.

cell biology↗

bioRxiv2018

Novel platelet-neutrophil interaction via activated αIIbβ3 mediates NETosis under flow

Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ib-dependent platelet priming induces integrin IIb{beta}3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet IIb{beta}3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated IIb{beta}3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil cross-talk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.\n\nSummaryPlatelets that are primed following interaction with von Willebrand factor under flow mediated direct interactions with neutrophils via activated platelet integrin, IIb{beta}3, and SLC44A2 on neutrophils. This interaction initiates signaling in a mechanosensitive manner that promotes neutrophil extracellular trap formation.

cell biology↗

bioRxiv2018

EphB2 and ERK signaling are required for heterotypic contact inhibition of locomotion to drive cell sorting

Interactions between different cell-types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesized to control population-wide behaviors during embryogenesis [1, 2]. However, our understanding of the signals that lead to cell-type specific repulsion, and the precise capacity of heterotypic CIL responses to drive emergent behaviors is lacking. Using a new in vitro model of heterotypic CIL between epithelial and mesenchymal cells, we show that fibrosarcoma cells (HT1080), but not fibroblasts (NIH3T3), are actively repelled by epithelial cells in culture. We show that knocking down EphB2 in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. Furthermore, this heterotypic interaction requires ERK activation, downstream of EphB2 signaling. We also examine the population-wide effects when these various cell combinations, and their specific heterotypic CIL responses, are allowed to interact in culture. Mixtures of fibrosarcoma and epithelial cells - unlike fibroblasts and epithelial cells - lead to complete sorting and segregation of the two populations, and inhibiting their distinct CIL response by knocking down EphB2 or ERK in fibrosarcoma cells disrupts this emergent sorting behavior. Our understanding of the mechanisms underlying developmental behaviors such as cell sorting is lacking as predominant sorting hypotheses, such as differential adhesion, have recently been found inadequate in predicting the sorting of mesenchymal cells [3, 4]. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations during embryogenesis.

cell biology↗

bioRxiv2018

The prokaryotic SPHINX 1.8 REP protein is tissue-specific and expressed in human germline cells

Small circular DNAs of 1.8 and 2.4kb were initially discovered in highly infectious CJD and scrapie particles from mammalian brain and cultured cells. Surprisingly, these protected cytoplasmic \"SPHINX\" DNAs contained replication (REP) initiation sequences resembling those of Acinetobacter phage viruses. An antibody was generated against a REP peptide encoded by the SPHINX 1.8 ORF that was not present in mammals. It bound to a 41kd \"spx1\" protein on Western blots. Cytologically, spx1 concentrated in spinal cord synapses and pancreatic islet, but not exocrine cells. We hypothesized that circular SPHINX DNAs are ancient symbiotic elements that can participate in functional differentiation and neurodegeneration. Cell and tissue specific patterns of spx1 expression shown below implicate somatic cell-to-cell communication and differentiation functions that would favor conservation of SPHINX 1.8 in evolution. Remarkably, primary human oocytes and spermatogonia, but not mature sperm, displayed intense cytoplasmic spx1 signals that underscore the maternal inheritance of SPHINX 1.8. These findings should encourage investigations of unexplored networks of incorporated environmental infectious agents that can be key actors in progressive neurodegeneration, immunity and cancer.

cell biology↗

bioRxiv2018

Accurate transmission of IGF-I into AKT signaling activity in individual cells

Cells sense and respond to their environment by activating distinct intracellular signaling pathways, however an individual cells ability to faithfully transmit and discriminate environmental signals is thought to be limited. To assess the fidelity of signal transmission in the PI3K-AKT signaling pathway, we first developed an optimized genetically encoded sensor that had an increased dynamic range and reduced variation under basal conditions. We then used this reporter to track responses to varying doses of IGF-I in live cells and found that signaling responses from individual cells overlapped across a wide range of IGF-I doses, suggesting limited transmission accuracy. However, further analysis of individual cell traces revealed that responses were constant over time without stochastic fluctuations. We devised a new information theoretic approach to calculate the channel capacity using variance of the single cell time course data--rather than population-level variance as has been previously used--and predicted that cells were capable of discriminating multiple growth factor doses. We validated these predictions by tracking individual cell responses to multiple IGF-I doses and found that cells can accurately distinguish at least four different IGF-I concentrations, as demonstrated by their distinct responses. Furthermore, we found a similar discriminatory ability to pathway inhibition, as assessed by responses to the PI3K inhibitor alpelisib. Our studies indicate that cells can faithfully transmit an IGF-I input into a down-stream signaling response and that heterogeneous responses result from variation in the input-output relation across the population. These observations reveal the importance of viewing each cell as having its own communication channel and underscore the importance of understanding responses at the single cell level.

cell biology↗

bioRxiv2018

Nuclear pore density controls heterochromatin reorganization during senescence

Oncogene induced senescence (OIS) is a cell cycle arrest program triggered by oncogenic signalling. An important characteristic of OIS is activation of the senescence associated secretory phenotype (SASP)1 which can reinforce cell cycle arrest, lead to paracrine senescence but also promote tumour progression2-4. Concomitant with cell cycle arrest and the SASP activation, OIS cells undergo a striking nuclear chromatin reorganization, with loss of heterochromatin from the nuclear periphery and the appearance of internal senescence-associated heterochromatin foci (SAHF)5. The mechanisms by which SAHF are formed, and their role in cell cycle arrest and expression of the SASP, remain poorly understood. Here we show that nuclear pore density increases during OIS and is responsible for SAHF formation. In particular, we show that the nucleoporin TPR is required for both SAHF formation and maintenance. The TPR-induced loss of SAHF does not affect cell cycle arrest but completely abrogates the SASP. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and in senescence, which uncouples the cell cycle arrest from the SASP.

cell biology↗

bioRxiv2018

EIPR1 controls dense-core vesicle cargo sorting and EARP complex localization in insulinoma cells

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargos including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting protein EIPR-1 as proteins important for controlling levels of DCV cargos in C. elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargos such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

cell biology↗

bioRxiv2018

Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling

Enteroendocrine cells (EECs) produce hormones that regulate food absorption, insulin secretion and appetite. Both EECs and their peptide products are foci of drug discovery programmes for diabetes and obesity. We compared the human and mouse EEC transcriptome and peptidome to validate mouse as a model of the human enteroendocrine axis. We present the first RNA sequencing analysis of human EECs, and demonstrate strong correlation with mouse, although with outliers including some low abundance G-protein coupled receptors. Liquid chromatography mass spectrometry (LC-MS) identified peptide hormone gradients along the human and mouse gut that should enhance progress in gut physiology and therapeutics.

cell biology↗