Volume 51, Issue 6, Pages 819-828 (September 2013) Proteasomes Activate Aggresome Disassembly and Clearance by Producing Unanchored Ubiquitin Chains  Rui Hao, Priyaanka Nanduri, Yanhua Rao, R. Scott Panichelli, Akihiro Ito, Minoru Yoshida, Tso-Pang Yao  Molecular Cell  Volume 51, Issue 6, Pages 819-828 (September 2013) DOI: 10.1016/j.molcel.2013.08.016 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 HDAC6 Is Required for Aggresome Deaggregation and Clearance A549 cells were treated with MG132 (5 μM) for 24 hr to induce aggresome formation followed by MG132-free medium for the indicated times. Aggresomes were identified by staining with an antibody against ubiquitin (ub, clone FK1). (A) Ubiquitin immunostaining (red) indicates that the large aggresomes (0 hr) were deaggregated (12 hr) before final clearance (24 hr). Bottom, zoomed areas (white squares). Nuclei were stained with DAPI (blue). The status of aggresomes at different time points after MG132 washout was quantified in the histogram. (B) Representative images of HDAC6 (green) in relationship to the aggresome (red) by double immunostaining during MG132 washout. Bottom, zoomed areas (white squares). Note that protein aggregates are no longer positive for HDAC6 after 12 hr washout (middle). (C) A549 cells were treated with an HDAC6-selective inhibitor, Tubastatin A (TBSA, 10 μM), pan-HDAC inhibitor, Trichostatin A (TSA, 1 μM), or class I HDAC inhibitors, MS275 (10 μM), or sodium butyrate (NaBut, 1 mM) during MG132 washout. The presence of aggresome was analyzed and quantified as described in (A). (D) Representative images of HDAC6 and the aggresome (ub) in TBSA-treated cells. For (A) and (C), three independent experiments were quantified. Error bars show ± SEM. The scale bar represents 25 μm. See also Figure S1. Molecular Cell 2013 51, 819-828DOI: (10.1016/j.molcel.2013.08.016) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Poh1 Facilitates Aggresome Clearance by Producing Unanchored Ubiquitin Chains (A) Immunopurified FLAG-HDAC6 produced in HEK 293T cells was incubated with unanchored ubiquitin chains (K63-linked Ub 1-7) or ubiquitinated protein (ub-protein, see the Experimental Procedures for details). The input and bound fractions were analyzed by immunoblotting with the indicated antibodies. (B) A549 cells were transfected with control or Poh1-specific siRNA and treated with MG132 (5 μM, 24 hr) in order to induce aggresome or followed by MG132 washout as indicated. Aggresomes (marked by arrows) were detected and quantified as described in Figure 1A. Values represent the mean ± SEM (n = 3). (C) Poh1 knockdown (KD) cells were treated with MG132 to induce aggresomes, and MG132 washout was performed in the presence of DMSO or 2.5 μM of nocodazole (Noc.) for 24 hr. Cells containing aggresomes were quantified and averaged from three independent experiments in the histogram. Note that nocodazole treatment disrupted microtubule networks (α-tubulin, green) but had no effect on aggresome clearance. (D) A549 cells stably expressing shRNA-resistant wild-type (WT) or catalytically inactive (CI) H113/115A mutant Poh1 were infected with Poh1-shRNA lentivirus. The percentage of cells containing an aggresome after MG132 washout was analyzed and quantified as described in (B). (E) Poh1 KD cells were pretreated with MG132 to induce aggresome formation. Cells were microinjected with indicated ubiquitin species or BSA mixed with fluorescence-conjugated dextran 3 hr after MG132 was removed. The presence of aggresomes was analyzed 21 hr postinjection. Injected cells were identified by dextran (green, top) and marked by white dotted lines (bottom). Aggresomes were identified by staining with an antibody specific for K48-linked ubiquitin (red, clone Apu2) and marked by white arrowheads in injected cells. Note that only Poh1 KD cells injected with the K63-linked ubiquitin chains do not contain aggresomes. Noninjected cells retained aggresomes under all conditions (yellow arrowheads) and served as an internal control. Right, the quantification from three independent experiments. In each experiment, 50 to 100 injected cells were scored. p value is indicated in the figure. Error bars indicate ± SEM. (F) Control and Poh1 KD A549 cells were treated with 5 μM MG132 for 24 hr or with MG132 followed by 24 hr washout in the presence or absence of 10 mM 3-MA, as indicated. Detergent-insoluble fractions were isolated from whole-cell lysates and resolved by SDS-PAGE followed by immunoblotting with a K48- or K63-specific ubiquitin antibodies, as indicated. Actin was used as a loading control. See also Figures S2, S3, S4, and S8. Molecular Cell 2013 51, 819-828DOI: (10.1016/j.molcel.2013.08.016) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 HDAC6 Binds Unanchored Ubiquitin Chains during Aggresome Clearance in a Poh1-Dependent Manner (A) Representative image of HDAC6 and aggresome (ub) in Poh1 KD cells 24 hr after MG132 washout. (B) FLAG-HDAC6 was immunoprecipitated under the following conditions: (1) no treatment, (2) 5 μM MG132 for 24 hr, (3) 12 hr MG132 washout, and (4) 24 hr MG132 washout. The immune complexes were subjected to heating, and the eluates were treated with isopeptidase T (IsoT) as indicated (see the Experimental Procedures for details). Samples were analyzed by immunoblotting with antibodies for ubiquitin or HDAC6. Note that MG132 washout (condition 2 versus 3) led to a decrease in total ubiquitinated protein levels in whole-cell lysate (WCL) but an increase in HDAC6-associated free ubiquitin chains. (C) Free ubiquitin chains associated with HDAC6 in WT and Poh1 KD cells were assessed 12 hr after MG132 washout. Note that Poh1 KD led to the accumulation of total ubiquitinated proteins (left) but a reduction of HDAC6-associated unanchored ubiquitin chains (right). (D) Free ubiquitin chains released from HDAC6-IP components were sequentially immunoblotted with a pan-ubiquitin antibody (pan-ub), K63-specific ubiquitin antibody (K63-ub), and K48-specific ubiquitin antibody (K48-ub). Recombinant K63- or K48-linked polyubiquitin chains were loaded onto the same gel in order to validate the specificity of linkage-specific ubiquitin antibodies. See also Figure S5. Molecular Cell 2013 51, 819-828DOI: (10.1016/j.molcel.2013.08.016) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Free Ubiquitin Chains Regulate HDAC6 Activity (A) Immunopurified FLAG-HDAC6 WT, H215A/H610A CI, and various point mutants were incubated with free ubiquitin chains as indicated. The bound fractions were detected by immunoblotting with an ubiquitin antibody. (B) Top, filter-trap analysis of SDS-insoluble ubiquitinated aggregates accumulated in WT, HDAC6 KO, and KO MEFs stably expressing human WT HDAC6 or W1182A mutant subject to 24 hr MG132 washout. The relative ubiquitin signal intensity was quantified and is presented in parenthesis under each genotype where HDAC6 KO MEFs was set at 100. Bottom, whole-cell extracts from indicated cell lines were immunoblotted with antibodies for human HDAC6 (hHDAC6), mouse HDAC6 (mHDAC6), and actin. (C) Cortactin, the acetyltransferase CBP, and WT or HDAC6 mutant plasmids were cotransfected into HEK 293T cells as indicated. Cortactin was immunoprecipitated and immunoblotted with an antibody for acetylated (Ac) cortactin or total cortactin. The relative cortactin-acetylation level (Ac-cortactin/cortactin) was quantified by scanning densitometry and is presented in the right panel. Error bars indicate ± SD (n = 3). The statistical significance was assessed with two-way ANOVA analysis with Dunnett’s test. ∗p < 0.05. See also Figure S6. Molecular Cell 2013 51, 819-828DOI: (10.1016/j.molcel.2013.08.016) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Deaggregation of the Aggresome Requires the Actinomyosin System A549 cells were pretreated with MG132 (5 μM) for 24 hr to induce aggresome formation. (A) Representative images of the aggresome (ubiquitin, green) and F-actin (red, Rhodamine-Phalloidin) 12 hr after MG132 washout in cells with indicated treatment. In the panels of F-actin, the location of the aggresome is marked by an arrowhead. Right, cells exhibiting F-actin punctae around the aggresome were quantified from three experiments. Error bars indicate ± SEM. ∗p < 0.01. (B) Representative staining of F-actin and a deaggregated aggresome 12 hr after MG132 washout. Right, the zoomed areas (white squares). (C) A549 cells transfected with the indicated siRNA were imaged 0 or 24 hr after MG132 washout. Aggresomes (arrows) were detected with an antibody against ubiquitin (red), and the percentage of cells retained an aggresome was quantified from three independent experiments. Error bars show ± SEM. (D) Costaining of HDAC6 (green) and ubiquitin (red) in cells expressing indicated siRNA 24 hr after MG132 washout. Arrows mark aggresomes. Right, the quantification of aggresomes that are positive for HDAC6 (n = 3). Error bars show ± SEM. ∗p < 0.01. See also Figure S7. Molecular Cell 2013 51, 819-828DOI: (10.1016/j.molcel.2013.08.016) Copyright © 2013 Elsevier Inc. Terms and Conditions