Figure 6.

Parkin inclusion bodies display aggresome-like characteristics. (A–H) Parkin inclusions cause disruption of γ-tubulin localization. COS-7 cells were transfected with FLAG-Parkin (A–C and E–G) in the presence (E–H) or absence (A–D) of MG132. Cells were stained with affinity-purified rabbit anti-Parkin (red) and mouse γ-tubulin (1:1000) (green) antibodies. Staining of untransfected cells confirmed aggregation of γ-tubulin only occurs in cells overexpressing Parkin (D and H). Red staining in D and H represents 4,6-diamidino-2-phenylindole staining of the nucleus. Arrowheads indicate γ-tubulin localization. (I–N) Partial colocalization of Parkin and vimentin near the centrosome. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and anti-vimentin (1:40) antibodies (green) in the presence (L–N) or absence (I–K) of MG132. Arrow indicates region of colocalization in N. (O–Q) Acetylated α-tubulin localizes to Parkin inclusions. Cells overexpressing Parkin were stained with affinity-purified rabbit anti-Parkin (red) and mouse anti-acetylated α-tubulin (1:2000) antibodies (green). Panels show untreated (O), MG132-treated (P), or combined MG132 and nocodazole-treated (Q) cells. Arrows indicate colocalization within the inclusion in P and Q. The asterisk (^*) indicates an untransfected cell with intact acetylated α-tubulin staining in Q. Overlays of each set include 4,6-diamidino-2-phenylindole staining (blue) (C, G, K, and N) to identify the nucleus. Mitochondria localize with Parkin inclusions. In R and S, Parkin expression is indicated by the green fluorescence and the mitochondria were labeled using MitoTracker (used at 300 nM; red fluorescence). With the exception of (R and S), which were fixed with 4% (wt/vol) paraformaldehyde, all cells were fixed with methanol. Bars, 20 μm.