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

To confirm the result that GRASP depletion reduces cell migration, we performed a Transwell assay using WT and GRASP-KO HeLa cells (Isaji et al., 2006) . In this assay, the same number of cells was seeded in the inserts with serum-free medium, and full growth medium with serum was added in the lower chamber as a chemoattractant. After a 20 h incubation, cells on the lower side of the membrane were fixed, stained with crystal violet, and imaged. The results showed that the migration rate was significantly reduced by the knockout of GRASP55 or GRASP65, while double deletion of both GRASPs had a more severe effect (Figure 4 , A-C). In the control, 184 ± 3 cells migrated to the lower side of the membrane per field of view, while GRASP55, GRASP65, and double-knockout cells had 94 ± 15, 100 ± 15, and 59 ± 28 cells migrate to the bottom side, respectively ( Figure 4C ). Exogenous expression of GFP-tagged GRASP65 or GRASP55 rescued the migration defect in GRASP-KO cells ( Figure 4D and Supplemental Figure 3 ). On the basis of the results from the wound-healing and Transwell assays, we conclude that GRASP depletion results in decreased cell migration and invasion.