GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of a5ß1 integrin

Ahat, Erpan; Xiang, Yi; Zhang, Xiaoyan; Bekier, Michael E.; Wang, Yanzhuang

The fact that Golgi unstacking reduces cell adhesion and migration (Figures 1-4) suggests that it may affect cell adhesion molecules such as integrin, as shown in Figure 3C . Therefore, we examined the effects of GRASP depletion on the protein level of several integrins that are known to be expressed in HeLa cells, including α1, α2, α3, α5, αV, α6, β1, β3, and β5 (Bergman et al., 1995; Liu et al., 2009; Scheffer et al., 2013; Wan et al., 2014; Ratcliffe et al., 2016) , by Western blot. As shown in Figure 5A , α1, αV, and α6 levels did not change in GRASP-KO cells compared with WT HeLa. α3 Integrin was reduced only in GRASP65 KO cells, while the β3 level was slightly reduced only in GRASP55 KO cells. Consistent with the results shown in The reduced migration of GRASP55-KO and GRASP65-KO cells was rescued by expressing GRASP55-GFP and GRASP65-GFP, respectively, but not by GFP alone. Results are presented as mean ± SEM; statistical analysis was performed by comparison with WT control (ctrl) using Student's t test. *, p < 0.05; **, p < 0.01. (C) Western blot of HeLa cells transfected with indicated constructs. α5-Integrin heavy chain (α5 integrin HC), GRASP55, GRASP65, GFP, and actin were blotted. The reduced protein levels of α5 integrin in GRASP55-KO and GRASP65-KO cells were rescued by expressing GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone. Figure 3C , both α5 and β1 integrins were robustly reduced in GRASP single-or double-knockout cells, suggesting that Golgi unstacking reduces cell adhesion by decreasing the α5β1-integrin level.