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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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Dominant-negative SNARE domains inhibit β1 integrin exocytosis. CHO-K1 cells were transiently transfected for 21 h with GFP-tagged constructs of truncated syntaxin13 (Syn13cyto)[D-F], VAMP3 (VAMP3cyto)[G-I], VAMP4 (VAMP4cyto) or SNAP23 (SNAP23CΔ9 - 15 h transfection) [J-L]. Untransfected control cells are shown in A-C. HeLa cells were transfected with a SNAP23 shRNA construct (SN23 shRNA) for 72 h. Cells were incubated with β1 integrin antibody in serum free media for 3 h to allowing internalization of the label. Then the cells were lifted with trypsin, removing remaining surface label, and were plated on 20 μg/mL FN for 10 min (A, D, G, J,), 20 min (B, E, H, K,) or 45 min (C, F, I, L). For exocytosis assays, cells were fixed and any surface exposed labeled-integrin was stained with AlexaFluor 594 secondary antibody (A-L, data for 20 min of adhesion shown in N). Insets show expression of GFP-tagged constructs (D-L). Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm. (M and N) Integrin immunofluorescent staining at 20 min. was quantified in micrographs using ImageJ software. (M) To assess endocytosis of labeled integrin, cells were permeablized with 0.2% Trition X-100 in PBS before staining with AlexaFluor 594 secondary antibody (percent of non-transfected control). (N) Labeled integrin detected on the cell surface in non-permeabilized cells (percent of non-transfected control). Means ± SEM from three independent experiments (at least 20 cells per experiment) are shown in M and N.
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Figure 4: Dominant-negative SNARE domains inhibit β1 integrin exocytosis. CHO-K1 cells were transiently transfected for 21 h with GFP-tagged constructs of truncated syntaxin13 (Syn13cyto)[D-F], VAMP3 (VAMP3cyto)[G-I], VAMP4 (VAMP4cyto) or SNAP23 (SNAP23CΔ9 - 15 h transfection) [J-L]. Untransfected control cells are shown in A-C. HeLa cells were transfected with a SNAP23 shRNA construct (SN23 shRNA) for 72 h. Cells were incubated with β1 integrin antibody in serum free media for 3 h to allowing internalization of the label. Then the cells were lifted with trypsin, removing remaining surface label, and were plated on 20 μg/mL FN for 10 min (A, D, G, J,), 20 min (B, E, H, K,) or 45 min (C, F, I, L). For exocytosis assays, cells were fixed and any surface exposed labeled-integrin was stained with AlexaFluor 594 secondary antibody (A-L, data for 20 min of adhesion shown in N). Insets show expression of GFP-tagged constructs (D-L). Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm. (M and N) Integrin immunofluorescent staining at 20 min. was quantified in micrographs using ImageJ software. (M) To assess endocytosis of labeled integrin, cells were permeablized with 0.2% Trition X-100 in PBS before staining with AlexaFluor 594 secondary antibody (percent of non-transfected control). (N) Labeled integrin detected on the cell surface in non-permeabilized cells (percent of non-transfected control). Means ± SEM from three independent experiments (at least 20 cells per experiment) are shown in M and N.

Mentions: In previous studies, we have demonstrated, using TeTx-LC, that VAMP3 is required for trafficking of β1 integrin from the recycling endosome to the plasma membrane and that this inhibition in traffic is a possible explanation for reduced spreading [16]. Here, we examined whether inhibition with the syntaxin13cyto, VAMP3cyto, SNAP23CΔ9, VAMP4cyto and the SNAP23 shRNA constructs also prevents exocytosis of β1 integrin. Integrin exocytosis was monitored by allowing internalized antibody-labeled β1 integrin to traffic to the plasma membrane during adhesion to FN. Internalization of label was confirmed by permeablization and staining with fluorescently labeled secondary antibody. Inhibiting SNARE function with the SNARE constructs or shRNA did not inhibit endocytosis of labeled integrin relative to controls (Fig. 4M). In non-transfected control cells, at 10 min, β1 integrin was visible on the cell surface and became more prominent at 20 min (Fig. 4A and 4B, respectively). At 45 min, the β1 integrin signal became more diffuse as the integrin was redistributed on the cell surface as well as internalized again (Fig. 4C). Transfection of cells with empty vector did not alter integrin trafficking (data not shown). Consistent with previous experiments using TeTx-LC [16], at 10 min VAMP3cyto-expressing cells show no labeled β1 integrin on their surface (Fig. 4D). After 20 and 45 min a modest amount of integrin is visible (Fig. 4E and 4F, respectively). In cells expressing syntaxin13cyto, little internalized β1 integrin is delivered to the cell surface at 10 min (Fig. 4G), while at 20 and 45 min time points only a modest amount of integrin has reached the surface (Fig. 4H and 4I, respectively). Cells expressing SNAP23CΔ9 show little or no labeled integrin on their surface at all time points examined (Fig. 4J-L). The surface integrin staining was quantified for the 20 min time point of three independent experiments and is shown in Fig. 4N. When VAMP3, syntaxin13 or SNAP23 were inhibited the exocytosis of labeled integrin was decreased by 60%. Similar results were seen when SNAP23 was knocked-down using an shRNA construct (Fig. 4N). By contrast, expression of VAMP4cyto slightly increased the appearance of label at the surface (Fig. 4N).


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)

Dominant-negative SNARE domains inhibit β1 integrin exocytosis. CHO-K1 cells were transiently transfected for 21 h with GFP-tagged constructs of truncated syntaxin13 (Syn13cyto)[D-F], VAMP3 (VAMP3cyto)[G-I], VAMP4 (VAMP4cyto) or SNAP23 (SNAP23CΔ9 - 15 h transfection) [J-L]. Untransfected control cells are shown in A-C. HeLa cells were transfected with a SNAP23 shRNA construct (SN23 shRNA) for 72 h. Cells were incubated with β1 integrin antibody in serum free media for 3 h to allowing internalization of the label. Then the cells were lifted with trypsin, removing remaining surface label, and were plated on 20 μg/mL FN for 10 min (A, D, G, J,), 20 min (B, E, H, K,) or 45 min (C, F, I, L). For exocytosis assays, cells were fixed and any surface exposed labeled-integrin was stained with AlexaFluor 594 secondary antibody (A-L, data for 20 min of adhesion shown in N). Insets show expression of GFP-tagged constructs (D-L). Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm. (M and N) Integrin immunofluorescent staining at 20 min. was quantified in micrographs using ImageJ software. (M) To assess endocytosis of labeled integrin, cells were permeablized with 0.2% Trition X-100 in PBS before staining with AlexaFluor 594 secondary antibody (percent of non-transfected control). (N) Labeled integrin detected on the cell surface in non-permeabilized cells (percent of non-transfected control). Means ± SEM from three independent experiments (at least 20 cells per experiment) are shown in M and N.
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Figure 4: Dominant-negative SNARE domains inhibit β1 integrin exocytosis. CHO-K1 cells were transiently transfected for 21 h with GFP-tagged constructs of truncated syntaxin13 (Syn13cyto)[D-F], VAMP3 (VAMP3cyto)[G-I], VAMP4 (VAMP4cyto) or SNAP23 (SNAP23CΔ9 - 15 h transfection) [J-L]. Untransfected control cells are shown in A-C. HeLa cells were transfected with a SNAP23 shRNA construct (SN23 shRNA) for 72 h. Cells were incubated with β1 integrin antibody in serum free media for 3 h to allowing internalization of the label. Then the cells were lifted with trypsin, removing remaining surface label, and were plated on 20 μg/mL FN for 10 min (A, D, G, J,), 20 min (B, E, H, K,) or 45 min (C, F, I, L). For exocytosis assays, cells were fixed and any surface exposed labeled-integrin was stained with AlexaFluor 594 secondary antibody (A-L, data for 20 min of adhesion shown in N). Insets show expression of GFP-tagged constructs (D-L). Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm. (M and N) Integrin immunofluorescent staining at 20 min. was quantified in micrographs using ImageJ software. (M) To assess endocytosis of labeled integrin, cells were permeablized with 0.2% Trition X-100 in PBS before staining with AlexaFluor 594 secondary antibody (percent of non-transfected control). (N) Labeled integrin detected on the cell surface in non-permeabilized cells (percent of non-transfected control). Means ± SEM from three independent experiments (at least 20 cells per experiment) are shown in M and N.
Mentions: In previous studies, we have demonstrated, using TeTx-LC, that VAMP3 is required for trafficking of β1 integrin from the recycling endosome to the plasma membrane and that this inhibition in traffic is a possible explanation for reduced spreading [16]. Here, we examined whether inhibition with the syntaxin13cyto, VAMP3cyto, SNAP23CΔ9, VAMP4cyto and the SNAP23 shRNA constructs also prevents exocytosis of β1 integrin. Integrin exocytosis was monitored by allowing internalized antibody-labeled β1 integrin to traffic to the plasma membrane during adhesion to FN. Internalization of label was confirmed by permeablization and staining with fluorescently labeled secondary antibody. Inhibiting SNARE function with the SNARE constructs or shRNA did not inhibit endocytosis of labeled integrin relative to controls (Fig. 4M). In non-transfected control cells, at 10 min, β1 integrin was visible on the cell surface and became more prominent at 20 min (Fig. 4A and 4B, respectively). At 45 min, the β1 integrin signal became more diffuse as the integrin was redistributed on the cell surface as well as internalized again (Fig. 4C). Transfection of cells with empty vector did not alter integrin trafficking (data not shown). Consistent with previous experiments using TeTx-LC [16], at 10 min VAMP3cyto-expressing cells show no labeled β1 integrin on their surface (Fig. 4D). After 20 and 45 min a modest amount of integrin is visible (Fig. 4E and 4F, respectively). In cells expressing syntaxin13cyto, little internalized β1 integrin is delivered to the cell surface at 10 min (Fig. 4G), while at 20 and 45 min time points only a modest amount of integrin has reached the surface (Fig. 4H and 4I, respectively). Cells expressing SNAP23CΔ9 show little or no labeled integrin on their surface at all time points examined (Fig. 4J-L). The surface integrin staining was quantified for the 20 min time point of three independent experiments and is shown in Fig. 4N. When VAMP3, syntaxin13 or SNAP23 were inhibited the exocytosis of labeled integrin was decreased by 60%. Similar results were seen when SNAP23 was knocked-down using an shRNA construct (Fig. 4N). By contrast, expression of VAMP4cyto slightly increased the appearance of label at the surface (Fig. 4N).

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