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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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Inhibition of SNAREs reduces the rate of lamellipodium protrusion. Transfected CHO-K1 cells were plated in serum-free media on fibronetin for 90 min and monitored in real-time. (A) Representative control (a) and SNAP23CΔ9 (b) cells are shown at initial acquisition (0 min) and after 90 min (90 min) in the top panels. Kymographs (a' and b') in lower panels represent progression of the cell edge along the lines indicated with the cell edge highlighted. (B) The protrusion and retraction rate of the cell edge was determined from kymographs using ImageJ software. Data is presented as the mean velocity of immediate edge movement from at least two cell edges from at least 3 cells (when possible the most protrusive regions of the cell were measured).
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Figure 3: Inhibition of SNAREs reduces the rate of lamellipodium protrusion. Transfected CHO-K1 cells were plated in serum-free media on fibronetin for 90 min and monitored in real-time. (A) Representative control (a) and SNAP23CΔ9 (b) cells are shown at initial acquisition (0 min) and after 90 min (90 min) in the top panels. Kymographs (a' and b') in lower panels represent progression of the cell edge along the lines indicated with the cell edge highlighted. (B) The protrusion and retraction rate of the cell edge was determined from kymographs using ImageJ software. Data is presented as the mean velocity of immediate edge movement from at least two cell edges from at least 3 cells (when possible the most protrusive regions of the cell were measured).

Mentions: The observation that blocking VAMP3, SNAP23 or syntaxin13 function led to an impairment of lamellipodium extension is consistent with two possible explanations. It is possible that cells with impaired SNARE function: 1) cannot extend lamellipodia efficiently; or 2) can extend lamellipodia, but the lamellipodia cannot form stable adhesive contacts with the substratum and therefore are retracted. To assess these two possibilities, lamellipodium protrusion and retraction were examined in real time from cells plated on fibronection for 90 minutes. Kymographs of cellular spreading were generated, and the rate and duration of protrusions and retractions were quantified. Representative kymographs are shown in Fig. 3A and the data is summarized in Fig. 3B. In support of a model where SNARE inhibition impairs lamellipodium protrusion, it was found that cells expressing syntaxin13cyto, VAMP3cyto and SNAP23CΔ9 did form lamellipodia, but did so at a significantly slower rate than control cells (p ≤ 0.05). Lamellipodia from control cells extended at a mean rate of 5.6 ± 0.4 μm/min, while syntaxin13cyto and VAMP3cyto cells protruded at a mean rate of 4.0 ± 0.1 and 3.8 ± 0.6 μm/min, respectively. Similar to the spreading assay above, SNAP23CΔ9 proved to be the most potent inhibitor of extension, with SNAP23CΔ9 cells protruding at a mean rate of 2.7 ± 0.5 μm/min, and VAMP4cyto only had a modest effect (mean protrusion rate of 4.8 ± 0.6 μm/min). SNARE inhibition also inhibited retraction velocity compared to control cells, though the differences where not as substantial as seen with protrusion velocity (Fig. 3B).


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)

Inhibition of SNAREs reduces the rate of lamellipodium protrusion. Transfected CHO-K1 cells were plated in serum-free media on fibronetin for 90 min and monitored in real-time. (A) Representative control (a) and SNAP23CΔ9 (b) cells are shown at initial acquisition (0 min) and after 90 min (90 min) in the top panels. Kymographs (a' and b') in lower panels represent progression of the cell edge along the lines indicated with the cell edge highlighted. (B) The protrusion and retraction rate of the cell edge was determined from kymographs using ImageJ software. Data is presented as the mean velocity of immediate edge movement from at least two cell edges from at least 3 cells (when possible the most protrusive regions of the cell were measured).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Inhibition of SNAREs reduces the rate of lamellipodium protrusion. Transfected CHO-K1 cells were plated in serum-free media on fibronetin for 90 min and monitored in real-time. (A) Representative control (a) and SNAP23CΔ9 (b) cells are shown at initial acquisition (0 min) and after 90 min (90 min) in the top panels. Kymographs (a' and b') in lower panels represent progression of the cell edge along the lines indicated with the cell edge highlighted. (B) The protrusion and retraction rate of the cell edge was determined from kymographs using ImageJ software. Data is presented as the mean velocity of immediate edge movement from at least two cell edges from at least 3 cells (when possible the most protrusive regions of the cell were measured).
Mentions: The observation that blocking VAMP3, SNAP23 or syntaxin13 function led to an impairment of lamellipodium extension is consistent with two possible explanations. It is possible that cells with impaired SNARE function: 1) cannot extend lamellipodia efficiently; or 2) can extend lamellipodia, but the lamellipodia cannot form stable adhesive contacts with the substratum and therefore are retracted. To assess these two possibilities, lamellipodium protrusion and retraction were examined in real time from cells plated on fibronection for 90 minutes. Kymographs of cellular spreading were generated, and the rate and duration of protrusions and retractions were quantified. Representative kymographs are shown in Fig. 3A and the data is summarized in Fig. 3B. In support of a model where SNARE inhibition impairs lamellipodium protrusion, it was found that cells expressing syntaxin13cyto, VAMP3cyto and SNAP23CΔ9 did form lamellipodia, but did so at a significantly slower rate than control cells (p ≤ 0.05). Lamellipodia from control cells extended at a mean rate of 5.6 ± 0.4 μm/min, while syntaxin13cyto and VAMP3cyto cells protruded at a mean rate of 4.0 ± 0.1 and 3.8 ± 0.6 μm/min, respectively. Similar to the spreading assay above, SNAP23CΔ9 proved to be the most potent inhibitor of extension, with SNAP23CΔ9 cells protruding at a mean rate of 2.7 ± 0.5 μm/min, and VAMP4cyto only had a modest effect (mean protrusion rate of 4.8 ± 0.6 μm/min). SNARE inhibition also inhibited retraction velocity compared to control cells, though the differences where not as substantial as seen with protrusion velocity (Fig. 3B).

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