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 determine the effect of Golgi unstacking on cell migration, we first used a well-established wound-healing assay using the human breast cancer cell line MDA-MB-231 (Liang et al., 2007) . Both GRASP55 and GRASP65 could be readily depleted by siRNA treatment in this cell line ( Figure 2A ). Seventy-two hours after siRNA transfection, fully confluent MDA-MB-231 cells were starved in serum-free medium for 24 h. A scratch was made in the confluent layer using a 200-µl pipette tip, and the cells were cultured in complete growth medium for another 20 h to allow the cells to migrate into the wounded region. When examined under a microscope, cells that had been transfected with control siRNA almost completely covered the wound area, while GRASP-depleted cells only covered a portion of the wound region ( Figure 2B ). Control siRNA-treated cells migrated ∼39 ± 1 µm/h, while cells depleted of GRASP55, GRASP65, or both migrated at 33 ± 1, 25 ± 2, and 19 ± 2 µm/h, respectively, significantly slower than the control cells ( Figure 2C ). The same wound-healing assay was done with GRASP-KO HeLa cells, and consistent results were obtained (Figure 3 and Supplemental Figure 2 ). Cells depleted of GRASP55, GRASP65, or both migrated at 22 ± 2, 16 ± 2, and 15 ± 2 µm/h, respectively, significantly slower than the 32 ± 3 µm/h of wild-type (WT) cells (Supplemental Figure 2 ). This defect was rescued by the expression of GFP-tagged GRASP65 or GRASP55, but not GFP alone ( Figure 3 , A-C). Interestingly, the level of the cell adhesion molecule α5 integrin appeared to be lower in GRASP-KO cells and was rescued by the expression of GRASP55-GFP and GRASP65-GFP in the corresponding knockout cells ( Figure 3C , lanes 6 and 7 vs. lanes 4 and 5).