SNARE-mediated trafficking of alpha5beta1 integrin is required for spreading in CHO cells.
In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of alpha5beta1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.
Skalski
Michael
M
Department of Molecular and Cellular Biology, University of Guleph, Guelph, Ont., Canada N1G 2W1.
Coppolino
Marc G
MG
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D039081:Integrin alpha5beta1; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins