TRIM50 Promotes Aggresome Formation and Clearance Figure 1. TRIM50 bodies colocalize into aggresome by a Hdac6-dependent way. (A) SH-SY5Y cells were stained with anti-TRIM50 antibody. (B) SH-SY5Y cells were stained with anti-TRIM50 and anti-HDAC6 antibodies. The cells were treated with 25 mM MG132 for 6 h (d,e,f). (C) Hdac6 wild type and Hdac6 deficient mouse fibroblasts were transfected with EGFP-Trim50 followed by treatment with 25 mM MG132 for 6 h where indicated, and immunostained with FK2 antibody. doi:10.1371/journal.pone.0040440.g001 TRIM50 determines the accumulation of both p62 and HDCA6 into an insoluble protein aggregate fraction. Results TRIM50 Localizes to Cytoplasmic Bodies We previously reported that ectopically expressed TRIM50 localizes mainly into discrete cytoplasmic punctuate structures heterogeneous in size and shape, with the intact central region of the protein (B-Box and Coiled-Coil domains) indispensable for the proper localization [15]. To rule out that the observed pattern was due to TRIM50 overexpression, we showed that also the endogenous TRIM50 localizes in diffuse cytoplasmic round bodies in human neuroblastoma-derived SH-SY5Y cell lines (Figure 1A). Ectopically expressed TRIM50 cytoplasmic bodies did not associate with known cellular compartments and markers includ- ing trans- and cis-Golgi, endosomes, caveolae, vesicles, lysosomes, cytosckeletal structures, peroxisomes, stress granules, and P-bodies (Figure S1). Using live microscopy, we showed that ectopically expressed TRIM50 cytoplasmic bodies are highly motile structures varying in size and shape that exhibit multidirectional short and fast jumping movements and able to assemble larger cytoplasmic bodies from smaller particles (Figure S2A–B, and Movie S1). To gain insight into the dynamics of TRIM50 bodies, we performed Fluorescence Recovery After Photobleaching (FRAP) [17]. Our analysis revealed that the fluorescence of a photobleached cytoplasmic body significantly recovers within the 2 min of the time-period experiment (Fig. S2C, and Movie S2). The FRAP data confirm that TRIM50 cytoplasmic bodies are dynamic, rapidly exchanging between different cytoplasmic regions, and promptly turned over. Further characterization of TRIM50 bodies was achieved by Correlative Light-Electron Microscopy (CLEM). This analysis revealed that the fluorescent bodies corresponded to heteroge- neous in morphology TRIM50-containing protein aggregates, confirming their tendency to self-associate into larger structures (Figure S2D). TRIM50 Associates with Aggresome We asked whether TRIM50 bodies associate with aggresome. In SH-SY5Y cells, treated with the proteasome inhibitor MG132 and stained with FK2, which recognizes polyubiquitinated proteins, endogenous TRIM50 concentrated close to a perinuclear structure whose morphology and localization resemble that of aggresome (Figure S3A, d–f). To further investigate the possible link between TRIM50 and aggresome, we used HDAC6, an established aggresome marker [18]. We found that both endog- enous and transfected TRIM50 partially located with HDAC6 under proteasome inhibition (Figure 1B, d–f; Figure S3B, d–f). This cellular localization does not depend on the E3-ligase activity of the RING domain of TRIM50 as a mutant lacking the RING domain retains the ability to localize to aggresome (Figure S4A). In accordance with the central role of retrograde microtu- bule-dependent transport in the formation of aggresome, nocodazole treatment of SH-SY5Y cells prevented the localiza- tion of TRIM50 to aggresome (data not shown). In agreement we demonstrated that TRIM50 interacts with Tubulin beta 2B class IIb (Tubb2b) (NM_178012.4), a microtubules component (Figure S3C). Together these findings suggest that TRIM50 bodies may represent aggresome precursors that in response to proteasome inhibition move towards aggresome by a microtu- bule dependent transport. To assess whether HDAC6 is required for the proper localization of TRIM50, we performed immunofluorescence assays in Hdac6 deficient mouse fibroblasts [19]. We found that in Hdac6 wild-type cells, Trim50 bodies localize within FK2- ubiquitin-containing aggresomes upon MG132 treatment (Figure 1C, d–f). Conversely, when MG132 was added to Hdac6 knock out cells Trim50 bodies were unable to form whole aggresome, although they still continued to partially colocalize with ubiquitinated aggregates (Figure 1C, j–l). These results indicate that HDAC6 is required for the proper localization of TRIM50 bodies and of ubiquitinated proteins within the aggresome. We then investigated whether the observed TRIM50-HDAC6 colocalization results also in their physical interaction. As no anti-TRIM50 antibodies are actually effective for immunoprecipitation assays, we generated a HEK293 cell line that stably expresses a FLAG-tagged TRIM50 (hereafter referred to as FLAG-TRIM50#3). As shown in Figure 2A, TRIM50 interacts with endogenous HDAC6; an interaction that strength- ens in response to MG132 treatment (compare lane 1 to lane 2 in Figure 2A). TRIM50 Interacts with p62 Proteomics assays were designed to identify novel TRIM50 partners. TRIM50 complexes were isolated by immunoprecipita- tion of total protein lysate from FLAG-TRIM50#3 cells and individual protein components were solved and identified by nano LC-MS/MS. Among the putative TRIM50 interactors, we focused on p62 (also known as Sequestosome 1/p62), because of its involvement in the formation of protein aggregates [20], its role as shuttling factor for the delivery of polyubiquitinated substrates to the proteasome [21], and for emerging central importance at the intersection of proteasome and autophagy pathways [22]. First we assessed whether TRIM50 and p62 self associate. FLAG- TRIM50#3 cells was transfected with a plasmid that expresses Myc-tagged p62. Total cell lysates were then immunoprecipitated with an anti-FLAG and immunoblotted with an anti-Myc specific antibody. An anti-Myc reactive band was exclusively precipitated in the presence of FLAG-TRIM50 (Figure 2C, left side). Consistently, we detected FLAG-TRIM50 in protein lysates immunoprecipitated with an anti-Myc and immunoblotted with an anti-FLAG antibody (Figure 2C, right side), substantiating the interaction between TRIM50 and p62. Next, to map the TRIM50 domain(s) involved in p62 interaction, we used a set of FLAG-TRIM50 mutants [15] (Figure 2B). Upon co-transfection with Myc-p62 and coimmuno- precipitation with an anti-FLAG, we found that both coiled coil domains of TRIM50 are required for the efficient binding of p62 (Figure 2D). Likewise, to define the p62 interaction region, we performed co-immunoprecipitation and GST pull down assays using p62 deletion mutants (Figure 3A). As reported in Figure 3B and 3C we narrowed the p62 interaction region between aminoacids 302–370, a region that includes the LC3 binding region [23]. PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40440