scholarly journals The tomosyn homologue, Sro7, is a direct effector of the Rab GTPase, Sec4, in post-Golgi vesicle tethering

2018 ◽  
Vol 29 (12) ◽  
pp. 1476-1486 ◽  
Author(s):  
Guendalina Rossi ◽  
Kelly Watson ◽  
Wade Kennedy ◽  
Patrick Brennwald
Keyword(s):  
Plasma Membrane ◽  
Simple Model ◽  
Genetic Analysis ◽  
Rab Gtpase ◽  
Vesicle Tethering ◽  
Effector Pathways ◽  
Rab Effector

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

scholarly journals Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

1997 ◽  
Vol 185 (3) ◽  
pp. 579-582 ◽  
Author(s):  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Simonetta Falzoni ◽  
Stefania Hanau ◽  
Francesco Di  Virgilio
Keyword(s):  
Plasma Membrane ◽  
P2x7 Receptor ◽  
Purinergic Receptor ◽  
Present Report ◽  
Physiological Role ◽  
Bacterial Endotoxin ◽  
Microglial Cells

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.

Multinucleated rat alveolar macrophages express functional receptors for calcitonin

1991 ◽  
Vol 261 (6) ◽  
pp. F1026-F1032 ◽  
Author(s):  
A. Vignery ◽  
M. J. Raymond ◽  
H. Y. Qian ◽  
F. Wang ◽  
S. A. Rosenzweig
Keyword(s):  
Plasma Membrane ◽  
Alveolar Macrophages ◽  
Giant Cells ◽  
Mononuclear Phagocytes ◽  
Inflammatory Reactions ◽  
Calcitonin Gene ◽  
Novel Model

The fusion of mononuclear phagocytes occurs spontaneously in vivo and leads to the differentiation of either multinucleated giant cells or osteoclasts in chronic inflammatory sites or in bone, respectively. Although osteoclasts are responsible for resorbing bone, the functional role of giant cells in chronic inflammatory reactions and tumors remains poorly understood. We recently reported that the plasma membrane of multinucleated macrophages is, like that of osteoclasts, enriched in Na-K-adenosinetriphosphatases (ATPases). We also observed that the localization of their Na-K-ATPases is restricted to the nonadherent domain of the plasma membrane of cells both in vivo and in vitro, thus imposing a functional polarity on their organization. By following this observation, we wished to investigate whether these cells also expressed, like osteoclasts, functional receptors for calcitonin (CT). To this end, alveolar macrophages were fused in vitro, and both their structural and functional association with CT was analyzed and compared with those of mononucleated peritoneal and alveolar macrophages. Evidence is presented that multinucleated alveolar macrophages express a high copy number of functional receptors for CT. Our results also indicate that alveolar macrophages, much like peritoneal, express functional receptors for calcitonin gene-related peptide. It is suggested that multinucleated rat alveolar macrophages offer a novel model system to study CT receptors and that calcitonin may control local immune reactions where giant cells differentiate.

Role of ESCRT component HD-PTP/PTPN23 in cancer

2017 ◽  
Vol 45 (3) ◽  
pp. 845-854 ◽  
Author(s):  
Marie-Claude Gingras ◽  
Jalal M. Kazan ◽  
Arnim Pause
Keyword(s):  
Plasma Membrane ◽  
Cancer Susceptibility ◽  
Tumour Suppressor ◽  
Tumour Suppressor Genes ◽  
Surface Receptors ◽  
Endosomal Sorting ◽  
Bona Fide

Sustained cellular signalling originated from the receptors located at the plasma membrane is widely associated with cancer susceptibility. Endosomal sorting and degradation of the cell surface receptors is therefore crucial to preventing chronic downstream signalling and tumorigenesis. Since the Endosomal Sorting Complexes Required for Transport (ESCRT) controls these processes, ESCRT components were proposed to act as tumour suppressor genes. However, the bona fide role of ESCRT components in tumorigenesis has not been clearly demonstrated. The ESCRT member HD-PTP/PTPN23 was recently identified as a novel haplo-insufficient tumour suppressor in vitro and in vivo, in mice and humans. In this mini-review, we outline the role of the ESCRT components in cancer and summarize the functions of HD-PTP/PTPN23 in tumorigenesis.

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

scholarly journals Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1β activation on macrophages

2020 ◽  
Vol 79 (11) ◽  
pp. 1506-1514
Author(s):  
Felix Renaudin ◽  
Lucie Orliaguet ◽  
Florence Castelli ◽  
François Fenaille ◽  
Aurelie Prignon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Aerobic Glycolysis ◽  
Metabolic Reprogramming ◽  
Metabolic Phenotype ◽  
Glucose Transporter 1 ◽  
Gout Flare

ObjectiveMacrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1β-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1β-induced microcrystal response.MethodsBriefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1β production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition.ResultsWe observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1β production, and microcrystal inflammation in vivo.ConclusionIn conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1β response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

scholarly journals 14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3947-3955 ◽  
Author(s):  
Sandrine Roy ◽  
Robert A. McPherson ◽  
Ann Apolloni ◽  
Jun Yan ◽  
Annette Lane ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Specific Activity ◽  
Membrane Recruitment ◽  
Oncogenic Ras ◽  
Epidermal Growth

ABSTRACT 14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

scholarly journals Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

1998 ◽  
Vol 143 (4) ◽  
pp. 1129-1141 ◽  
Author(s):  
Daqing W. Hartwell ◽  
Tanya N. Mayadas ◽  
Gaëtan Berger ◽  
Paul S. Frenette ◽  
Helen Rayburn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Inflammatory Responses ◽  
Cytoplasmic Domain ◽  
Leukocyte Rolling ◽  
Storage Pool ◽  
Storage Granules

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

scholarly journals The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6165
Author(s):  
Liubov Shimolina ◽  
Alexander Gulin ◽  
Nadezhda Ignatova ◽  
Irina Druzhkova ◽  
Margarita Gubina ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasma Membrane ◽  
Lipid Profile ◽  
Cancer Cells ◽  
Fluorescence Lifetime Imaging ◽  
Molecular Rotor ◽  
Membrane Viscosity

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.

scholarly journals The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

2003 ◽  
Vol 162 (4) ◽  
pp. 635-646 ◽  
Author(s):  
Xiang-Dong Gao ◽  
Stefan Albert ◽  
Serguei E. Tcheperegine ◽  
Christopher G. Burd ◽  
Dieter Gallwitz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Gtpase Activating Protein ◽  
Actin Organization ◽  
Polarized Secretion ◽  
Actin Patch ◽  
Actin Cables

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80–100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.

scholarly journals TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11

2015 ◽  
Vol 112 (14) ◽  
pp. 4346-4351 ◽  
Author(s):  
Mario Pinar ◽  
Herbert N. Arst ◽  
Areti Pantazopoulou ◽  
Víctor G. Tagua ◽  
Vivian de los Ríos ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Missense Mutations ◽  
Nucleotide Exchange ◽  
Protein Particle ◽  
Oligomeric Complex ◽  
Specific Association ◽  
Substantial Extent

The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabORAB1 resides in the Golgi, RabERAB11 localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabERAB11, but not RabORAB1, immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabERAB11 in vivo. hypA1ts rapidly shifts RabERAB11, but not RabORAB1, to the cytosol, consistent with HypATrs120 being specifically required for RabERAB11 activation. Missense mutations rescuing hypA1ts at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabORAB1 did not, establishing that the essential role of TRAPPII is facilitating RabERAB11 nucleotide exchange. In vitro, TRAPPII purified with HypATrs120-S-tag accelerates nucleotide exchange on RabERAB11 and, paradoxically, to a lesser yet substantial extent, on RabORAB1. Evidence obtained by exploiting hypA1-mediated destabilization of HypATrs120/HypCTrs130/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabORAB1, which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypATrs120 colocalizes with RabERAB11, arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi–to–post-Golgi transition.

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