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Shared and specific functions of Arfs 1-5 at the Golgi revealed by systematic knockouts.

ABSTRACT: ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.

SUBMITTER: Pennauer M

PROVIDER: S-EPMC8579194 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Shared and specific functions of Arfs 1-5 at the golgi revealed by systematic knockouts

Project description:DP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4, and Arf4+5 could not be deleted simultaneously. Class I Arfs are non-essential and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.

2021-11-02 | PXD028846 | Pride

Arfaptins are localized to the trans-Golgi by interaction with Arl1, but not Arfs.

Project description:Arfaptins (arfaptin-1 and arfaptin-2/POR1) were originally identified as binding partners of the Arf small GTPases. Both proteins contain a BAR (Bin/Amphiphysin/Rvs) domain, which participates in membrane deformation. Here we show that arfaptins associate with trans-Golgi membranes. Unexpectedly, Arl1 (Arf-like 1), but not Arfs, determines the trans-Golgi association of arfaptins. We also demonstrate that arfaptins interact with Arl1 through their BAR domain-containing region and compete for Arl1 binding with golgin-97 and golgin-245/p230, both of which also bind to Arl1 through their GRIP (golgin-97/RanBP2/Imh1p/p230) domains. However, arfaptins and these golgins show only limited colocalization at the trans-Golgi. Time-lapse imaging of cells overexpressing fluorescent protein-tagged arfaptins and golgin-97 reveals that arfaptins, but not golgin-97, are included in vesicular and tubular structures emanating from the Golgi region. These observations indicate that arfaptins are recruited onto trans-Golgi membranes by interacting with Arl1, and capable of inducing membrane deformation via their BAR domains.

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Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1.

Project description:Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addition, loss of Arf x GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf x GTP, rather than the formation of an Arf x GDP x BFA x GBF1 complex.

| S-EPMC2488303 | biostudies-literature

SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

Project description:The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

| S-EPMC8684707 | biostudies-literature

Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.

Project description:In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

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Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses.

Project description:The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

| S-EPMC4935974 | biostudies-literature

Novel cardiovascular gene functions revealed via systematic phenotype prediction in zebrafish.

Project description:Comprehensive functional annotation of vertebrate genomes is fundamental to biological discovery. Reverse genetic screening has been highly useful for determination of gene function, but is untenable as a systematic approach in vertebrate model organisms given the number of surveyable genes and observable phenotypes. Unbiased prediction of gene-phenotype relationships offers a strategy to direct finite experimental resources towards likely phenotypes, thus maximizing de novo discovery of gene functions. Here we prioritized genes for phenotypic assay in zebrafish through machine learning, predicting the effect of loss of function of each of 15,106 zebrafish genes on 338 distinct embryonic anatomical processes. Focusing on cardiovascular phenotypes, the learning procedure predicted known knockdown and mutant phenotypes with high precision. In proof-of-concept studies we validated 16 high-confidence cardiac predictions using targeted morpholino knockdown and initial blinded phenotyping in embryonic zebrafish, confirming a significant enrichment for cardiac phenotypes as compared with morpholino controls. Subsequent detailed analyses of cardiac function confirmed these results, identifying novel physiological defects for 11 tested genes. Among these we identified tmem88a, a recently described attenuator of Wnt signaling, as a discrete regulator of the patterning of intercellular coupling in the zebrafish cardiac epithelium. Thus, we show that systematic prioritization in zebrafish can accelerate the pace of developmental gene function discovery.

| S-EPMC3865760 | biostudies-literature

Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells.

Project description:TRα1 and TRβ1, the two main thyroid hormone receptors in mammals, are transcription factors that share similar properties. However, their respective functions are very different. This functional divergence might be explained in two ways: it can reflect different expression patterns or result from different intrinsic properties of the receptors. We tested this second hypothesis by comparing the repertoires of 3,3',5-triiodo-L-thyronine (T3)-responsive genes of two neural cell lines, expressing either TRα1 or TRβ1. Using transcriptome analysis, we found that a substantial fraction of the T3 target genes display a marked preference for one of the two receptors. So when placed alone in identical situations, the two receptors have different repertoires of target genes. Chromatin occupancy analysis, performed at a genome-wide scale, revealed that TRα1 and TRβ1 cistromes were also different. However, receptor-selective regulation of T3 target genes did not result from receptor-selective chromatin occupancy of their promoter regions. We conclude that modification of TRα1 and TRβ1 intrinsic properties contributes in a large part to the divergent evolution of the receptors' function, at least during neurodevelopment.

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Genome-wide analysis of thyroid hormone receptors' shared and specific functions in neural cells

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2012-12-31 | E-GEOD-41727 | biostudies-arrayexpress

A newly identified myomegalin isoform functions in Golgi microtubule organization and ER-Golgi transport.

Project description:The Golgi of mammalian cells is known to be a major microtubule-organizing site that requires microtubules for its organization and protein trafficking. However, the mechanisms underlying the microtubule organization of the Golgi remain obscure. We used immunoprecipitation coupled with mass spectrometry to identify a widely expressed isoform of the poorly characterized muscle protein myomegalin. This newly identified isoform, myomegalin variant 8 (MMG8), localized predominantly to cis-Golgi networks by interacting with AKAP450 (also known as AKAP9), and this interaction with AKAP450 was required for the stability of both proteins. Disrupting MMG8 expression affected endoplasmic reticulum (ER)-to-Golgi trafficking and caused Golgi fragmentation. Furthermore, MMG8 associated with γ-tubulin complexes and with the microtubule plus-end tracking protein EB1 (also known as MAPRE1), and was required for the Golgi localization of these two molecules. On the Golgi, γ-tubulin complexes mediated microtubule nucleation, whereas EB1 functioned in ER-to-Golgi trafficking. These results indicate that MMG8 participates in Golgi microtubule organization and thereby plays a crucial role in the organization and function of the Golgi.

| S-EPMC6518325 | biostudies-literature

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