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 proper glycosylation and localization of cell surface proteins are important for cell adhesion and migration. We observed that, when examined under a phase-contrast microscope, HeLa cells depleted of GRASP65 or both GRASPs spread less well on the dish, and they also formed clusters and appeared to be rounder compared with cells transfected with control or GRASP55 small interfering RNA (siRNA) (Figure 1, C and D vs. A and B, and E) . To further assess the effect of GRASP depletion on cell adhesion, we detached the cells using EDTA and placed the cells on fibronectin-coated plates. After incubation with serum-free medium at 37°C for 30 min, the attached cells were counted. Compared with the control, in which 55 ± 4% of cells were attached to the dish, only 38 ± 1% of GRASP55depleted cells and 31 ± 3% of GRASP65-depleted cells bound to the dish ( Figure 1F ). When both GRASP55 and GRASP65 were depleted, the percentage of attached cells was further reduced to 22 ± 4% ( Figure 1F ). This effect was rescued by the exogenous expression of green fluorescent protein (GFP)-tagged GRASP65 or GRASP55, but not GFP alone (Figure 1 , G and H). Similar results were obtained in HeLa cells in which GRASPs were knocked out by CRISPR/Cas9-mediated genome editing (Supplemental Figure 1 ) (Bekier et al., 2017) . These results demonstrate that Golgi unstacking reduces cell adhesion.