Skalski Michael, Coppolino Marc G
Department of Molecular and Cellular Biology, University of Guleph, Guelph, Ont., Canada N1G 2W1.
Biochem Biophys Res Commun. 2005 Oct 7;335(4):1199-210. doi: 10.1016/j.bbrc.2005.07.195.
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.
在本研究中，我们检测了SNARE介导的膜转运在调节整合素定位中的作用，并定量评估了细胞铺展过程中SNARE功能的需求。使用破伤风毒素（TeTx-LC）的VAMP特异性催化轻链、N-乙基马来酰亚胺敏感融合蛋白（NSF）的显性负性形式（E329Q）和布雷菲德菌素A（BfA）抑制膜转运。用E329Q-NSF或TeTx-LC而非BfA抑制膜转运，可显著抑制CHO细胞在纤连蛋白上的铺展。通过共转染抗TeTx的细胞ubrevin/VAMP3，可在表达TeTx-LC的细胞中挽救铺展。E329Q-NSF作为SNARE功能的一般抑制剂，比TeTx-LC更有效地抑制细胞铺展，这表明破伤风毒素不敏感的SNARE有助于黏附。研究发现，E329Q-NSF阻止了α5β1整合素在细胞黏附过程中从含Rab11的中央区室转运至突出部位，而TeTx-LC则延迟了这种转运。这些结果与一种细胞黏附模型一致，该模型认为SNARE功能是细胞铺展过程中整合素转运的重要组成部分。
