FIGURE 6.

Localization of AHA1 in migrating cells. A, localization of HSP90α, HSP90β, RAB3GAP1, and AHA1 in PC3-MM2 cells. Wild type PC3-MM2 cells were fixed and stained for HSP90α and AHA1, RAB3GAP1, and AHA1 or HSP90β. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. The representative images show that AHA1 co-localizes with HSP90α and RAB3GAP1. RAB3GAP1 is a marker for secretory vesicles. The secretory vesicles containing AHA1 and HSP90α or RAB3GAP1 are marked with white arrows. HSP90β is localized in the cytoplasm and the cell cortex region. At least 25 images were processed for the co-localization assay. B, heat map of AHA1-GFP in control and GRP94 KD cells in a cell migration assay. Cell migration montage of the control and GRP94 KD cells transfected with AHA1-GFP plasmid. Fluorescent photomicrographs of the control and GRP94 KD cells were taken at 15-min intervals for 9 h of the cell migration/wound healing assay. Results are representative of three independent wound healing assays per cell line. Time stamp, hh:mm:ss; scale bar, 10 μm. The white arrows in the control cells indicate AHA1-GFP localization at the tip of the filopodia in the direction of the migration. The white arrows in the GRP94 KD cells also indicate the AHA1-GFP localization, but instead of filopodia, thick and short pseudopodia were observed throughout the cell membrane. C, localization of AHA1 in the control, GRP94 KD, and GRP94 revertant cells. Control, GRP94 KD, and GRP94 revertant cells were fixed and stained for AHA1 and DAPI (blue). Scale bar, 10 μm. The white arrows in the control cells indicate AHA1 localization in secretory vesicles, but the staining is evident in the endoplasmic reticulum in GRP94 KD cells. Fluorescent images are representative of three independent biological replicates.