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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 disrupt β1 integrin trafficking into the recycling endosome. CHO-K1 cells were transiently transfected for 22 h with GFP [A-D] or GFP-tagged constructs of truncated VAMP3 (V3cyto) [E-H], syntaxin13 (Syn13cyto) [I-L], SNAP23 (SNAP23CΔ9 - 15 h transfection) [M-P, A'-D'], GS15 (GS15cyto) [Q-T], or VAMP4 (V4cyto) [U-X]. Cells were labeled with anti-β1 integrin antibody in serum free medium for 1 h to allow internalization of integrin-antibody complexes. Cells were then washed in 0.2 M glycine, pH 2.5, fixed, and stained for Rab11 [C, G, K, O, S, W and C'] and the intracellular integrin-antibody complexes [B, F, J, N, R, and V], or Rab4 [B']. Overlays of the Rab11 and β1 integrin staining are shown in D, H, L, P, T and X, and Rab4 and Rab11 overlays are shown in D'. Arrows indicate conventional colocalization of β1 integrin in a central Rab11-containing compartment. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.
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Figure 5: Dominant-negative SNARE domains disrupt β1 integrin trafficking into the recycling endosome. CHO-K1 cells were transiently transfected for 22 h with GFP [A-D] or GFP-tagged constructs of truncated VAMP3 (V3cyto) [E-H], syntaxin13 (Syn13cyto) [I-L], SNAP23 (SNAP23CΔ9 - 15 h transfection) [M-P, A'-D'], GS15 (GS15cyto) [Q-T], or VAMP4 (V4cyto) [U-X]. Cells were labeled with anti-β1 integrin antibody in serum free medium for 1 h to allow internalization of integrin-antibody complexes. Cells were then washed in 0.2 M glycine, pH 2.5, fixed, and stained for Rab11 [C, G, K, O, S, W and C'] and the intracellular integrin-antibody complexes [B, F, J, N, R, and V], or Rab4 [B']. Overlays of the Rab11 and β1 integrin staining are shown in D, H, L, P, T and X, and Rab4 and Rab11 overlays are shown in D'. Arrows indicate conventional colocalization of β1 integrin in a central Rab11-containing compartment. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.

Mentions: Having shown that VAMP3, syntaxin13 and SNAP23 form a complex, and that inhibition of these SNAREs impairs integrin exocytosis, we sought to determine where within the cell β1 integrin was being trapped. CHO cells expressing dominant-negative SNARE domains were incubated with β1 integrin antibody for one hour followed by an acid wash to remove surface antibody. The subcellular localization of the internalized integrin and the endocytic compartment markers Rab11 and Rab4 was then assessed (Fig. 5 and Additional file 4). In control, GFP-transfected cells (Fig. 5A-D), a portion of the internalized integrin localizes in the cell periphery with the sorting endosome marker Rab4 (Additional file 4B-D). Another portion of β1 localizes in a concentrated, perinuclear compartment, overlapping with the recycling endosome marker Rab11 (Fig. 5B-D, see arrow in 5D). These distributions were also observed in non-transfected controls (data not shown). Strikingly, in cells expressing VAMP3cyto, syntaxin13cyto or SNAP23CΔ9, the peripheral localization of β1 was unaltered, but there was little or no perinuclear β1- or Rab11-containing compartment (Fig. 5E-P). In cells expressing the dominant-negative SNARE inhibitors, the concentrated, central compartment containing Rab11 appeared to be disrupted, becoming punctate and peripherally distributed (Fig. 5E-P, contrast with Fig. 5B-D). It is important to note, however, that the association of integrin with Rab11 was maintained, though the morphology and location of this compartment was dramatically altered. Further analysis revealed that expression of the mutant SNAREs had caused Rab11 to redistribute and extensively co-localize with the Rab4 compartment (Fig. 5A'-D'; SNAP23CΔ9 shown as representative images). The inhibition of this pathway does not appear to be specific to integrin trafficking as the VAMP3cyto, syntaxin13cyto and SNAP23CΔ9 also inhibit transferrin trafficking into a perinuclear compartment (Additional file 5). Expression of VAMP4cyto (Fig. 5Q-T) and GS15cyto (Fig. 5U-X) had no effect on β1 integrin or transferrin (Additional file 5G-H) trafficking into the perinuclear compartment. Neither VAMP4cyto nor GS15cyto altered the morphology of the recycling endosome to the degree that VAMP3cyto, syntaxin13cyto or SNAP23CΔ9 did (Fig. 5S and 5W).


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 disrupt β1 integrin trafficking into the recycling endosome. CHO-K1 cells were transiently transfected for 22 h with GFP [A-D] or GFP-tagged constructs of truncated VAMP3 (V3cyto) [E-H], syntaxin13 (Syn13cyto) [I-L], SNAP23 (SNAP23CΔ9 - 15 h transfection) [M-P, A'-D'], GS15 (GS15cyto) [Q-T], or VAMP4 (V4cyto) [U-X]. Cells were labeled with anti-β1 integrin antibody in serum free medium for 1 h to allow internalization of integrin-antibody complexes. Cells were then washed in 0.2 M glycine, pH 2.5, fixed, and stained for Rab11 [C, G, K, O, S, W and C'] and the intracellular integrin-antibody complexes [B, F, J, N, R, and V], or Rab4 [B']. Overlays of the Rab11 and β1 integrin staining are shown in D, H, L, P, T and X, and Rab4 and Rab11 overlays are shown in D'. Arrows indicate conventional colocalization of β1 integrin in a central Rab11-containing compartment. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2925818&req=5

Figure 5: Dominant-negative SNARE domains disrupt β1 integrin trafficking into the recycling endosome. CHO-K1 cells were transiently transfected for 22 h with GFP [A-D] or GFP-tagged constructs of truncated VAMP3 (V3cyto) [E-H], syntaxin13 (Syn13cyto) [I-L], SNAP23 (SNAP23CΔ9 - 15 h transfection) [M-P, A'-D'], GS15 (GS15cyto) [Q-T], or VAMP4 (V4cyto) [U-X]. Cells were labeled with anti-β1 integrin antibody in serum free medium for 1 h to allow internalization of integrin-antibody complexes. Cells were then washed in 0.2 M glycine, pH 2.5, fixed, and stained for Rab11 [C, G, K, O, S, W and C'] and the intracellular integrin-antibody complexes [B, F, J, N, R, and V], or Rab4 [B']. Overlays of the Rab11 and β1 integrin staining are shown in D, H, L, P, T and X, and Rab4 and Rab11 overlays are shown in D'. Arrows indicate conventional colocalization of β1 integrin in a central Rab11-containing compartment. Images are 3 D reconstructions of a z-series. Scale bar represents 10 μm.
Mentions: Having shown that VAMP3, syntaxin13 and SNAP23 form a complex, and that inhibition of these SNAREs impairs integrin exocytosis, we sought to determine where within the cell β1 integrin was being trapped. CHO cells expressing dominant-negative SNARE domains were incubated with β1 integrin antibody for one hour followed by an acid wash to remove surface antibody. The subcellular localization of the internalized integrin and the endocytic compartment markers Rab11 and Rab4 was then assessed (Fig. 5 and Additional file 4). In control, GFP-transfected cells (Fig. 5A-D), a portion of the internalized integrin localizes in the cell periphery with the sorting endosome marker Rab4 (Additional file 4B-D). Another portion of β1 localizes in a concentrated, perinuclear compartment, overlapping with the recycling endosome marker Rab11 (Fig. 5B-D, see arrow in 5D). These distributions were also observed in non-transfected controls (data not shown). Strikingly, in cells expressing VAMP3cyto, syntaxin13cyto or SNAP23CΔ9, the peripheral localization of β1 was unaltered, but there was little or no perinuclear β1- or Rab11-containing compartment (Fig. 5E-P). In cells expressing the dominant-negative SNARE inhibitors, the concentrated, central compartment containing Rab11 appeared to be disrupted, becoming punctate and peripherally distributed (Fig. 5E-P, contrast with Fig. 5B-D). It is important to note, however, that the association of integrin with Rab11 was maintained, though the morphology and location of this compartment was dramatically altered. Further analysis revealed that expression of the mutant SNAREs had caused Rab11 to redistribute and extensively co-localize with the Rab4 compartment (Fig. 5A'-D'; SNAP23CΔ9 shown as representative images). The inhibition of this pathway does not appear to be specific to integrin trafficking as the VAMP3cyto, syntaxin13cyto and SNAP23CΔ9 also inhibit transferrin trafficking into a perinuclear compartment (Additional file 5). Expression of VAMP4cyto (Fig. 5Q-T) and GS15cyto (Fig. 5U-X) had no effect on β1 integrin or transferrin (Additional file 5G-H) trafficking into the perinuclear compartment. Neither VAMP4cyto nor GS15cyto altered the morphology of the recycling endosome to the degree that VAMP3cyto, syntaxin13cyto or SNAP23CΔ9 did (Fig. 5S and 5W).

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