Parkin is an E3 ubiquitin ligase that is rapidly recruited to mitochondria in a PINK1-dependent manner in response to loss of mitochondrial membrane potential (Narendra et al., 2008, 2010). Parkin recruitment results in ubiquitination of mitochondria, followed by mitochondrial fission, clustering, and subsequent clearance of damaged mitochondria (Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010). To XAV-939 cell line address the hypothesis that VCP participates in the PINK1/Parkin pathway, we first assessed whether VCP is recruited

to mitochondria in response to depolarization. We used HeLa cells stably expressing YFP-Parkin (HeLa cells don’t show detectable endogenous Parkin expression, data not shown) and transfected plasmids expressing VCP-mCherry and mito-Cerulean (Cerulean fluorescent protein tagged with a mitochondrial targeting sequence). In untreated cells, Parkin and VCP were both distributed diffusely with no colocalization with mitochondria. However, 3 hr after treatment with the mitochondrial uncoupling agent carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), virtually all of the YFP-Parkin and VCP-mCherry signal colocalized with mito-Cerulean, illustrating recruitment to the mitochondria (Figure 3A). We also generated mouse embryonic fibroblasts (MEFs) stably expressing mito-Cerulean to enable monitoring

of mitochondria. These cells were cotransfected with plasmids selleck screening library else expressing mCherry-Parkin and VCP-EGFP, which as expected

were distributed diffusely throughout untreated cells. Within 3 hr of treatment with CCCP, mCherry-Parkin and VCP-EGFP signals were relocalized to mitochondria (Figure S3A). Notably, in the absence of exogenous Parkin we observed no recruitment of VCP to mitochondria in response to depolarization, suggesting that VCP recruitment is Parkin-dependent (Figure S3B). Entirely consistent with the results in HeLa cells and MEFs, we observed that VCP also was recruited to depolarized mitochondria in neuroblastoma-derived Sy5y cells (Figure S4) and myoblast-derived C2C12 cells (Figure S5). Thus, VCP is corecruited to depolarized mitochondria in concert with Parkin in a wide variety of cell types. To characterize mitochondrial recruitment of Parkin and VCP in detail, we performed dynamic imaging in HeLa cells expressing VCP-EGFP and mCherry-Parkin after treatment with CCCP. We consistently observed that recruitment of mCherry-Parkin to mitochondria preceded recruitment of VCP-EGFP (Figures 3B–3D and Movies S1 and S2). Indeed, in an analysis of 35 sequential movies of individual cells we found that Parkin relocalized to mitochondria approximately 20 min after CCCP treatment and that VCP followed Parkin approximately 15 min later (Figures 3C and 3D). We sought to examine VCP recruitment to mitochondria in a cell type that expresses endogenous Parkin.