FIGURE 4.

ARL6 is a bona fide centrosomal/basal body-associated protein that localizes independently of the microtubule network or the dynein molecular motor. A, endogenous ARL6, observed using the ARL6C antibody (red) in ciliated IMCD3 cells, is observed at peri-nuclear puncta that co-localize with γ-tubulin (green), a centrosomal/basal body marker (yellow in the merged image). Inset, 2× magnification of area shown. DNA is stained blue with 4′,6-diamidino-2-phenylindole. Arrowheads point to ARL6 localization to centrosomes. B, endogenous ARL6 (visualized using the ARL6N antibody in IMCD3 cells; red) remains associated with the centrosome (marked using an antibody against γ-tubulin; green) throughout the cell cycle (yellow signal in merged image). Note that the interphase cell is ciliated (stained with antibody against acetylated (acet) tubulin; green). C, nocodazole treatment, but not the control treatment (DMSO), for either 1 h or overnight (O/N), is sufficient to destroy the microtubule network in IMCD3 cells assayed by α-tubulin staining (green), but it has no effect on ARL6 (red) localization. Arrowheads point to ARL6 localized to centrosomes in cells treated with nocodazole overnight. D, inhibition of the molecular motor dynein is achieved through the overexpression of green fluorescent protein-tagged p50-dynamitin (green) shown by the ability to mislocalize PCM1 (right panel), a protein known to be dependent upon dynein for its centrosomal localization (note its dispersed staining in cells expressing p50-dynamitin compared with cells not expressing the dynein inhibitor). ARL6 (left panel) is not mislocalized when p50 dynamitin is overexpressed. Arrowheads point to the proper localization of ARL6 in cells expressing p50-dynamitin.