scholarly journals Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

2004 ◽  
Vol 15 (10) ◽  
pp. 4609-4621 ◽  
Author(s):  
Kelly Tedrick ◽  
Tim Trischuk ◽  
Richard Lehner ◽  
Gary Eitzen
Keyword(s):  
Membrane Fusion ◽  
Actin Binding ◽  
Membrane Microdomains ◽  
Growth Defect ◽  
Dependent Manner ◽  
Actin Remodeling ◽  
Vacuole Fusion ◽  
Ergosterol Synthesis

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Δ growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Δ mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Δ vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Δ strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Δ-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Δ strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.

scholarly journals RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Ying-Ying Liang ◽  
Xu-Bin Deng ◽  
Xian-Tao Lin ◽  
Li-Li Jiang ◽  
Xiao-Ting Huang ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Underlying Mechanism ◽  
Carcinoma Cells ◽  
Dependent Manner ◽  
Transcriptional Coactivator ◽  
Actin Remodeling ◽  
Aggressive Tumor ◽  
Subunit B

Abstract Nasopharyngeal carcinoma (NPC) is a highly aggressive tumor characterized by distant metastasis. Deletion or down-regulation of the tumor suppressor protein ras-association domain family protein1 isoform A (RASSF1A) has been confirmed to be a key event in NPC progression; however, little is known about the effects or underlying mechanism of RASSF1A on the malignant phenotype. In the present study, we observed that RASSF1A expression inhibited the malignant phenotypes of NPC cells. Stable silencing of RASSF1A in NPC cell lines induced self-renewal properties and tumorigenicity in vivo/in vitro and the acquisition of an invasive phenotype in vitro. Mechanistically, RASSF1A inactivated Yes-associated Protein 1 (YAP1), a transcriptional coactivator, through actin remodeling, which further contributed to Platelet Derived Growth Factor Subunit B (PDGFB) transcription inhibition. Treatment with ectopic PDGFB partially increased the malignancy of NPC cells with transient knockdown of YAP1. Collectively, these findings suggest that RASSF1A inhibits malignant phenotypes by repressing PDGFB expression in a YAP1-dependent manner. PDGFB may serve as a potential interest of therapeutic regulators in patients with metastatic NPC.

scholarly journals The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex

2005 ◽  
Vol 168 (3) ◽  
pp. 401-414 ◽  
Author(s):  
Tracy J. LaGrassa ◽  
Christian Ungermann
Keyword(s):  
Cellular Membrane ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Casein Kinase I ◽  
Functional Protein ◽  
Hypertonic Stress ◽  
Functional Studies ◽  
Vacuole Fusion

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.

SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Glen Pearce ◽  
Tatsiana Audzevich ◽  
Rolf Jessberger
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
B Cell ◽  
Cell Polarization ◽  
Actin Binding ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Interacting Protein

Abstract B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.

Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin

2007 ◽  
Vol 292 (4) ◽  
pp. C1323-C1330 ◽  
Author(s):  
Teresia M. Osborn ◽  
Claes Dahlgren ◽  
John H. Hartwig ◽  
Thomas P. Stossel
Keyword(s):  
Platelet Activating Factor ◽  
Protective Role ◽  
Actin Binding ◽  
Dependent Manner ◽  
Protective Effects ◽  
Concentration Dependent Manner ◽  
Blood Concentrations ◽  
Plasma Gelsolin

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin (pGSN). Blood concentrations of pGSN decrease in response to diverse tissue injuries. Depletion of pGSN to critical levels precedes and often predicts complications of injuries such as lung permeability changes and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. One proposed mechanism for pGSN's protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. However, no direct evidence in support of this hypothesis has been available. Here we show that recombinant pGSN modestly inhibited LPA-induced P-selectin upregulation by human platelets in the presence of albumin ( P < 0.0001). However, physiologically relevant pGSN concentrations inhibit platelet-activating factor (PAF)-mediated P-selectin expression by up to 77% ( P < 0.0001). pGSN also markedly inhibited PAF-induced superoxide anion (O2−) production of human peripheral neutrophils (PMN) in a concentration-dependent manner ( P < 0.0001). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O2− generation ( P = 0.024). Therefore, pGSN interferes with PAF- and LPA-induced cellular activation in vitro, suggesting a mechanism for the protective role of pGSN in vivo.

scholarly journals The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae

2012 ◽  
Vol 197 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Kamran Anwar ◽  
Robin W. Klemm ◽  
Amanda Condon ◽  
Katharina N. Severin ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Dependent Manner ◽  
In Cells

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER–ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.

scholarly journals Substrate priming enhances phosphorylation by the budding yeast kinases Kin1 and Kin2

2018 ◽  
Vol 293 (47) ◽  
pp. 18353-18364
Author(s):  
Grace R. Jeschke ◽  
Hua Jane Lou ◽  
Keith Weise ◽  
Charlotte I. Hammond ◽  
Mallory Demonch ◽  
...  
Keyword(s):  
Er Stress ◽  
Secretory Pathway ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Growth Defect ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Dependent Manner

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo. Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.

Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions

1998 ◽  
Vol 111 (12) ◽  
pp. 1695-1706 ◽  
Author(s):  
B. Constantin ◽  
K. Meerschaert ◽  
J. Vandekerckhove ◽  
J. Gettemans
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Morphology ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Binding ◽  
General Mechanism ◽  
Resting Cells ◽  
Dependent Manner

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

scholarly journals Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1–dependent ABL1 activation in endothelial cells

2014 ◽  
Vol 211 (6) ◽  
pp. 1167-1183 ◽  
Author(s):  
Claudio Raimondi ◽  
Alessandro Fantin ◽  
Anastasia Lampropoulou ◽  
Laura Denti ◽  
Anissa Chikh ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Dependent Manner ◽  
Actin Remodeling ◽  
Angiogenic Therapy ◽  
Neuropilin 1 ◽  
Molecule Inhibitor ◽  
Vessel Growth ◽  
Integrin Ligand

To enable new blood vessel growth, endothelial cells (ECs) express neuropilin 1 (NRP1), and NRP1 associates with the receptor tyrosine kinase VEGFR2 after binding the vascular endothelial growth factor A (VEGF) to enhance arteriogenesis. We report that NRP1 contributes to angiogenesis through a novel mechanism. In human and mouse ECs, the integrin ligand fibronectin (FN) stimulated actin remodeling and phosphorylation of the focal adhesion component paxillin (PXN) in a VEGF/VEGFR2-independent but NRP1-dependent manner. NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling. This complex promoted EC motility in vitro and during angiogenesis on FN substrates in vivo. Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins. The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth.

scholarly journals A ToxR-dependent role for the putative phosphoporin VCA1008 in bile salt resistance in Vibrio cholerae El Tor N16961

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3011-3020 ◽  
Author(s):  
Carolina Lage Goulart ◽  
Guilherme Garcia dos Santos ◽  
Livia Carvalho Barbosa ◽  
Letícia Miranda Santos Lery ◽  
Paulo Mascarello Bisch ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Sodium Deoxycholate ◽  
Salt Resistance ◽  
Growth Defect ◽  
Dependent Manner ◽  
Pandemic Strains ◽  
El Tor ◽  
First Time

The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (Pi) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low Pi supply in the presence of DOC than in the absence of the detergent. In the Pi-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.

scholarly journals In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

1999 ◽  
Vol 146 (4) ◽  
pp. 819-830 ◽  
Author(s):  
Evelyne Ferrary ◽  
Michel Cohen-Tannoudji ◽  
Gérard Pehau-Arnaudet ◽  
Alexandre Lapillonne ◽  
Rafika Athman ◽  
...  
Keyword(s):  
Cell Injury ◽  
Actin Binding ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Wild Type ◽  
Death Probability ◽  
Brush Borders

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

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