The Experts below are selected from a list of 930 Experts worldwide ranked by ideXlab platform
Pablo A Ortiz - One of the best experts on this subject based on the ideXlab platform.
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vesicle associated membrane protein 3 VAMP3 mediates constitutive trafficking of the renal co transporter nkcc2 in thick ascending limbs role in renal function and blood pressure
Journal of Biological Chemistry, 2016Co-Authors: Mariela Mendez, Paulo S Caceres, Pablo A Ortiz, Mohammed Ziaul HaqueAbstract:Abstract Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl- co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3 and they co-localize at the TAL apical surface. We observed that silencing VAMP3 in vivo blocks constitutive NKCC2 exocytic delivery, decreasing the amount of NKCC2 at the TAL apical surface. VAMP3 is not required for cAMP-stimulated NKCC2 exocytic delivery. Additionally, genetic deletion of VAMP3 in mice decreased total expression of NKCC2 in the TAL and lowered blood pressure. Consistent with these results, urinary excretion of water and electrolytes was higher in VAMP3 knockout mice, which produced more diluted urine. We conclude that VAMP3 interacts with NKCC2 and mediates its constitutive exocytic delivery to the apical surface. Additionally, VAMP3 is required for normal NKCC2 expression, renal function and blood pressure.
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decreased surface nkcc2 expression and increased urinary excretion of water and ions in VAMP3 knockout mice
The FASEB Journal, 2015Co-Authors: Paulo S Caceres, Pablo A Ortiz, Mohammed Ziaul HaqueAbstract:Trafficking of the NKCC2 cotransporter to the apical surface of thick ascending limbs (TAL) regulates excretion of urine, ions and blood pressure. We showed that vesicle fusion proteins VAMP2 and VAMP3 mediate NKCC2 exocytosis to the TAL plasma membrane. VAMP3 also mediates NKCC2 expression. We showed that VAMP3-/- mice have lower blood pressure in normal and low-Na diet. However, it is not know whether surface NKCC2 is decreased in VAMP3-/- mice and the contribution of VAMP3 to renal function is unclear. We hypothesize that VAMP3-/- mice have decreased surface NKCC2 expression and increased excretion of water and ions. First we studied VAMP3-/- kidney histology and we found no abnormalities. Next, we measured surface NKCC2 in TAL suspensions by surface biotinylation. Surface NKCC2 was decreased by 46 ± 4% compared to wt. We also observed 23% higher urine volume (wt= 1.3 ± 0.1 ml/day vs VAMP3-/- = 1.6 ± 0.2 ml/day) and lower urine osmolality (wt= 3433 ± 166 mOsm vs VAMP3-/- = 2924 ± 131 mOsm). UNa, UK and...
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vesicle associated membrane protein 2 vamp2 but not VAMP3 mediates camp stimulated trafficking of the renal na k 2cl co transporter nkcc2 in thick ascending limbs
Journal of Biological Chemistry, 2014Co-Authors: Mariela Mendez, Paulo S Caceres, Pablo A OrtizAbstract:In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
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Vesicle-associated membrane protein 2 (VAMP2) but Not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl- co-transporter NKCC2 in thick ascending limbs.
Journal of Biological Chemistry, 2014Co-Authors: Paulo S Caceres, Mariela Mendez, Pablo A OrtizAbstract:In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
Paulo S Caceres - One of the best experts on this subject based on the ideXlab platform.
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vesicle associated membrane protein 3 VAMP3 mediates constitutive trafficking of the renal co transporter nkcc2 in thick ascending limbs role in renal function and blood pressure
Journal of Biological Chemistry, 2016Co-Authors: Mariela Mendez, Paulo S Caceres, Pablo A Ortiz, Mohammed Ziaul HaqueAbstract:Abstract Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl- co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3 and they co-localize at the TAL apical surface. We observed that silencing VAMP3 in vivo blocks constitutive NKCC2 exocytic delivery, decreasing the amount of NKCC2 at the TAL apical surface. VAMP3 is not required for cAMP-stimulated NKCC2 exocytic delivery. Additionally, genetic deletion of VAMP3 in mice decreased total expression of NKCC2 in the TAL and lowered blood pressure. Consistent with these results, urinary excretion of water and electrolytes was higher in VAMP3 knockout mice, which produced more diluted urine. We conclude that VAMP3 interacts with NKCC2 and mediates its constitutive exocytic delivery to the apical surface. Additionally, VAMP3 is required for normal NKCC2 expression, renal function and blood pressure.
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decreased surface nkcc2 expression and increased urinary excretion of water and ions in VAMP3 knockout mice
The FASEB Journal, 2015Co-Authors: Paulo S Caceres, Pablo A Ortiz, Mohammed Ziaul HaqueAbstract:Trafficking of the NKCC2 cotransporter to the apical surface of thick ascending limbs (TAL) regulates excretion of urine, ions and blood pressure. We showed that vesicle fusion proteins VAMP2 and VAMP3 mediate NKCC2 exocytosis to the TAL plasma membrane. VAMP3 also mediates NKCC2 expression. We showed that VAMP3-/- mice have lower blood pressure in normal and low-Na diet. However, it is not know whether surface NKCC2 is decreased in VAMP3-/- mice and the contribution of VAMP3 to renal function is unclear. We hypothesize that VAMP3-/- mice have decreased surface NKCC2 expression and increased excretion of water and ions. First we studied VAMP3-/- kidney histology and we found no abnormalities. Next, we measured surface NKCC2 in TAL suspensions by surface biotinylation. Surface NKCC2 was decreased by 46 ± 4% compared to wt. We also observed 23% higher urine volume (wt= 1.3 ± 0.1 ml/day vs VAMP3-/- = 1.6 ± 0.2 ml/day) and lower urine osmolality (wt= 3433 ± 166 mOsm vs VAMP3-/- = 2924 ± 131 mOsm). UNa, UK and...
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vesicle associated membrane protein 2 vamp2 but not VAMP3 mediates camp stimulated trafficking of the renal na k 2cl co transporter nkcc2 in thick ascending limbs
Journal of Biological Chemistry, 2014Co-Authors: Mariela Mendez, Paulo S Caceres, Pablo A OrtizAbstract:In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
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Vesicle-associated membrane protein 2 (VAMP2) but Not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl- co-transporter NKCC2 in thick ascending limbs.
Journal of Biological Chemistry, 2014Co-Authors: Paulo S Caceres, Mariela Mendez, Pablo A OrtizAbstract:In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
Jeffrey E Pessin - One of the best experts on this subject based on the ideXlab platform.
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AQP2 exocytosis in the renal collecting duct -- involvement of SNARE isoforms and the regulatory role of Munc18b.
Journal of cell science, 2008Co-Authors: Giuseppe Procino, Jeffrey E Pessin, Claudia Barbieri, Grazia Tamma, Leonarda De Benedictis, Maria Svelto, Giovanna ValentiAbstract:Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.
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AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b
Journal of Cell Science, 2008Co-Authors: Giuseppe Procino, Jeffrey E Pessin, Claudia Barbieri, Grazia Tamma, Leonarda De Benedictis, Maria Svelto, Giovanna ValentiAbstract:Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.
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rate and extent of phagocytosis in macrophages lacking VAMP3
Journal of Leukocyte Biology, 2002Co-Authors: Leeann H Allen, Chunmei Yang, Jeffrey E PessinAbstract:During phagocytosis, macrophages rapidly internalize a substantial fraction of plasma membrane without a net loss of surface area, suggesting that membranes are targeted to the cell surface from intracellular sites. Nevertheless, a requirement for mobilization of specific membrane compartments has not been demonstrated. We used bone marrow-derived macrophages (BMM) from wild type and VAMP3 null mice to evaluate directly the requirement for this v-SNARE in phagocytosis of zymosan, IgG-beads, complement-opsonized particles, or latex microspheres. Regardless of the phagocytic receptor engaged or particle load, BMM lacking VAMP3 exhibited no phagocytic defects when assayed after 1 h at 37°C, and phagosome maturation was unimpaired as judged by acquisition of lamp-1. In contrast, at early time points (5–15 min), internalization of zymosan (but not other particles tested) was significantly slower in VAMP3 null BMM. These data indicate that VAMP3 modulates efficient uptake of zymosan, but is not absolutely required for phagocytosis in primary macrophages.
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VAMP3 null mice display normal constitutive insulin and exercise regulated vesicle trafficking
Molecular and Cellular Biology, 2001Co-Authors: Chunmei Yang, Silvia Mora, Jeffrey W Ryder, Kenneth J Coker, Polly Hansen, Leeann H Allen, Jeffrey E PessinAbstract:To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.
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syntaxin 4 vamp2 and or VAMP3 cellubrevin are functional target membrane and vesicle snap receptors for insulin stimulated glut4 translocation in adipocytes
Molecular and Cellular Biology, 1997Co-Authors: Ann Louise Olson, John B Knight, Jeffrey E PessinAbstract:Introduction of the cytoplasmic domain of syntaxin 4, using either recombinant vaccinia virus or single-cell microinjection, resulted in an inhibition of insulin-stimulated GLUT4 but not GLUT1 translocation to the plasma membrane. This was specific for syntaxin 4, since neither the expression of syntaxin 3 nor the expression of a syntaxin 4 mutant in which the vesicle-associated membrane protein (VAMP) binding site was deleted had any significant effect. Consistent with the requirement for a functional VAMP binding site, expression of the cytoplasmic domains of VAMP2 or VAMP3/cellubrevin also resulted in an inhibition of insulin-stimulated GLUT4 translocation. In addition, immunoprecipitation of the expressed syntaxin 4 cytoplasmic domain resulted in an insulin-stimulated increase in the coimmunoprecipitation of GLUT4-containing vesicles. Together, these data demonstrate that syntaxin 4, VAMP2, and/or VAMP3/cellubrevin can function as target membrane and vesicle SNAP receptors, respectively, for insulin-responsive GLUT4 translocation to the plasma membrane.
Rachael Z Murray - One of the best experts on this subject based on the ideXlab platform.
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VAMP3 regulates podosome organisation in macrophages and together with stx4 snap23 mediates adhesion cell spreading and persistent migration
Experimental Cell Research, 2011Co-Authors: Kelly J Veale, Carolin Offenhauser, Amanda C Stanley, Jennifer L Stow, Rachael Z MurrayAbstract:The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.
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VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration
Experimental Cell Research, 2011Co-Authors: Kelly J Veale, Carolin Offenhauser, Amanda C Stanley, Jennifer L Stow, Rachael Z MurrayAbstract:The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.
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Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration.
Traffic, 2010Co-Authors: Kelly J Veale, Carolin Offenhauser, Shane P. Whittaker, Ruby P. Estrella, Rachael Z MurrayAbstract:In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.
Chuan Hu - One of the best experts on this subject based on the ideXlab platform.
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regulation of integrin endocytic recycling and chemotactic cell migration by syntaxin 6 and VAMP3 interaction
Journal of Cell Science, 2012Co-Authors: Krista A Riggs, Nazarul Hasan, David M Humphrey, Christy Raleigh, Chris Nevitt, Deborah R Corbin, Chuan HuAbstract:Integrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor α3β1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a rate-limiting role in cell migration and integrin trafficking. In STX6-depleted cells, α3β1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in α3β1 integrin delivery to the cell surface, and endocytosed α3β1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.
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silencing of VAMP3 inhibits cell migration and integrin mediated adhesion
Biochemical and Biophysical Research Communications, 2009Co-Authors: Kevin Luftman, Nazarul Hasan, Deborah Hardee, Chuan HuAbstract:Abstract Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.
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membrane fusion by VAMP3 and plasma membrane t snares
Experimental Cell Research, 2007Co-Authors: Deborah Hardee, Chuan Hu, Fred L MinnearAbstract:Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.
