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Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways.

Skalski M, Yi Q, Kean MJ, Myers DW, Williams KC, Burtnik A, Coppolino MG - BMC Cell Biol. (2010)

Bottom Line: Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading.Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13.Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guleph, Guelph, ON N1G 2W1, Canada.

ABSTRACT

Background: Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling.

Results: We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling.

Conclusions: Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.

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Analysis of SNARE function in PMA-induced membrane ruffle formation. CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with anti-SNAP23 antibody, Alexa-488-conjugated secondary antibody (A, D) and rhodamine-phalloidin (B, E). A-C, control cell; D-F, PMA-treated cell. Some cross-reactive binding of the SNAP23 antibody in the nucleus is observed (A, D). C and F are overlays of panels A and B and D and E, respectively. Scale bar represents 10 μm. (G) CHO-K1 cells were transfected with GFP vector alone, GFP-tagged dominant-negative constructs of the indicated SNAREs, or GFP together with the light chain of tetanus toxin. HeLa cells were transfected for 72 h with GFP + PLOK-1 or GFP + vector containing SNAP23-targeting shRNA. Cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with rhodamine-phalloidin. Samples were then imaged by confocal microscopy and actin-rich ruffles on the dorsal region of the cells were quantified as described previously. Ruffle indices (expressed as mean +/- SEM) are shown from three independent experiments (more than 25 randomly selected transfected cells were analyzed per condition in each experiment). (H and I) CHO-K1 cells were treated as above, lysed, and VAMP4 (H) or VAMP3 (I) were immunoprecipitated. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for SNAP23, upper blots. Then membranes were stripped and reprobed for the precipitated SNARE, lower blots.
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Figure 7: Analysis of SNARE function in PMA-induced membrane ruffle formation. CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with anti-SNAP23 antibody, Alexa-488-conjugated secondary antibody (A, D) and rhodamine-phalloidin (B, E). A-C, control cell; D-F, PMA-treated cell. Some cross-reactive binding of the SNAP23 antibody in the nucleus is observed (A, D). C and F are overlays of panels A and B and D and E, respectively. Scale bar represents 10 μm. (G) CHO-K1 cells were transfected with GFP vector alone, GFP-tagged dominant-negative constructs of the indicated SNAREs, or GFP together with the light chain of tetanus toxin. HeLa cells were transfected for 72 h with GFP + PLOK-1 or GFP + vector containing SNAP23-targeting shRNA. Cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with rhodamine-phalloidin. Samples were then imaged by confocal microscopy and actin-rich ruffles on the dorsal region of the cells were quantified as described previously. Ruffle indices (expressed as mean +/- SEM) are shown from three independent experiments (more than 25 randomly selected transfected cells were analyzed per condition in each experiment). (H and I) CHO-K1 cells were treated as above, lysed, and VAMP4 (H) or VAMP3 (I) were immunoprecipitated. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for SNAP23, upper blots. Then membranes were stripped and reprobed for the precipitated SNARE, lower blots.

Mentions: In CHO cells, endogenous SNAP23 was detected in PMA-induced membrane ruffles (Fig. 7A-F). While this observation is consistent with a role for SNAP23 in ruffle formation, we sought to directly assess the function of SNAP23, and SNAREs with which it interacts, in the formation of ruffles. To quantify membrane ruffles, we have developed a novel and rigorous method to automatically define and measure F-actin-rich ruffle structures in confocal micrographs [31]. CHO cells were transfected with the inhibitory SNARE constructs described above and F-actin-based ruffle formation was quantified. As seen in Fig. 7G, expression of SNAP23CΔ9 substantially reduced ruffle formation - the ruffle index for GFP-expressing/PMA-treated cells was 3.62 ± 0.4 and that for SNAP23CΔ9-expressing/PMA-treated cells was 1.20 ± 0.21, a 67% decrease (Fig. 7G). Confirming the effect of SNAP23 inhibition on membrane ruffling, we observed that shRNA mediated knock-down of SNAP23 expression reduced ruffle formation in HeLa cells (ruffle index of 1.57 ± 0.3, compared to PLOK1 vector controls with a ruffle index of 3.51 ± 0.47). HeLa cells were used for these experiments due to their compatibility with our human sequence-specific shRNA construct.


Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways.

Skalski M, Yi Q, Kean MJ, Myers DW, Williams KC, Burtnik A, Coppolino MG - BMC Cell Biol. (2010)

Analysis of SNARE function in PMA-induced membrane ruffle formation. CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with anti-SNAP23 antibody, Alexa-488-conjugated secondary antibody (A, D) and rhodamine-phalloidin (B, E). A-C, control cell; D-F, PMA-treated cell. Some cross-reactive binding of the SNAP23 antibody in the nucleus is observed (A, D). C and F are overlays of panels A and B and D and E, respectively. Scale bar represents 10 μm. (G) CHO-K1 cells were transfected with GFP vector alone, GFP-tagged dominant-negative constructs of the indicated SNAREs, or GFP together with the light chain of tetanus toxin. HeLa cells were transfected for 72 h with GFP + PLOK-1 or GFP + vector containing SNAP23-targeting shRNA. Cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with rhodamine-phalloidin. Samples were then imaged by confocal microscopy and actin-rich ruffles on the dorsal region of the cells were quantified as described previously. Ruffle indices (expressed as mean +/- SEM) are shown from three independent experiments (more than 25 randomly selected transfected cells were analyzed per condition in each experiment). (H and I) CHO-K1 cells were treated as above, lysed, and VAMP4 (H) or VAMP3 (I) were immunoprecipitated. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for SNAP23, upper blots. Then membranes were stripped and reprobed for the precipitated SNARE, lower blots.
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Figure 7: Analysis of SNARE function in PMA-induced membrane ruffle formation. CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with anti-SNAP23 antibody, Alexa-488-conjugated secondary antibody (A, D) and rhodamine-phalloidin (B, E). A-C, control cell; D-F, PMA-treated cell. Some cross-reactive binding of the SNAP23 antibody in the nucleus is observed (A, D). C and F are overlays of panels A and B and D and E, respectively. Scale bar represents 10 μm. (G) CHO-K1 cells were transfected with GFP vector alone, GFP-tagged dominant-negative constructs of the indicated SNAREs, or GFP together with the light chain of tetanus toxin. HeLa cells were transfected for 72 h with GFP + PLOK-1 or GFP + vector containing SNAP23-targeting shRNA. Cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with rhodamine-phalloidin. Samples were then imaged by confocal microscopy and actin-rich ruffles on the dorsal region of the cells were quantified as described previously. Ruffle indices (expressed as mean +/- SEM) are shown from three independent experiments (more than 25 randomly selected transfected cells were analyzed per condition in each experiment). (H and I) CHO-K1 cells were treated as above, lysed, and VAMP4 (H) or VAMP3 (I) were immunoprecipitated. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for SNAP23, upper blots. Then membranes were stripped and reprobed for the precipitated SNARE, lower blots.
Mentions: In CHO cells, endogenous SNAP23 was detected in PMA-induced membrane ruffles (Fig. 7A-F). While this observation is consistent with a role for SNAP23 in ruffle formation, we sought to directly assess the function of SNAP23, and SNAREs with which it interacts, in the formation of ruffles. To quantify membrane ruffles, we have developed a novel and rigorous method to automatically define and measure F-actin-rich ruffle structures in confocal micrographs [31]. CHO cells were transfected with the inhibitory SNARE constructs described above and F-actin-based ruffle formation was quantified. As seen in Fig. 7G, expression of SNAP23CΔ9 substantially reduced ruffle formation - the ruffle index for GFP-expressing/PMA-treated cells was 3.62 ± 0.4 and that for SNAP23CΔ9-expressing/PMA-treated cells was 1.20 ± 0.21, a 67% decrease (Fig. 7G). Confirming the effect of SNAP23 inhibition on membrane ruffling, we observed that shRNA mediated knock-down of SNAP23 expression reduced ruffle formation in HeLa cells (ruffle index of 1.57 ± 0.3, compared to PLOK1 vector controls with a ruffle index of 3.51 ± 0.47). HeLa cells were used for these experiments due to their compatibility with our human sequence-specific shRNA construct.

Bottom Line: Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading.Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13.Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guleph, Guelph, ON N1G 2W1, Canada.

ABSTRACT

Background: Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling.

Results: We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling.

Conclusions: Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.

Show MeSH
Related in: MedlinePlus