HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.

(B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6.(C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm.(D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).(A) Wild‐type and HDAC6 KO MEFs were transfected with pcDNA, pcDNA‐HDAC6WT, HDAC6CD, or HDAC6ΔBuz, along with mCherry‐GFP‐LC3 as indicated. Yellow signals indicate non‐acidic autophagosomes and red signals indicate acidic autophagolysosomes. Scale bar, 10 μm.(B) The total number of yellow vesicles was quantified from three independent experiments (>12 cells each) and presented as the percentage of total mCherry‐GFP‐LC3 dots (red plus yellow) along with the standard deviation.(C)In vitro fusion assays. Autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC6−/− MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Supplementary Figure S3). Values are means+s.e. of the percentages of fusion from three independent experiments (more than 10 images per each experiment).(D) EM images of wild‐type and HDAC6 KO MEFs in normal growth conditions. Yellow arrows, autophagosomes; red arrows, autophagolysosomes; green arrowheads, multilamellar bodies.(E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.(A) Autophagosome-lysosome fusion is analysed in wild‐type and HDAC6 KO MEFs with or without starvation (6 h) using mCherry‐GFP‐LC3 as described in Figure 2A.(B) Wild‐type and HDAC6 KO MEFs were cultured in Hank's solution for 3 h followed by immunoblotting with an antibody for LC3, HDAC6, and GAPDH.(C) Long‐lived protein degradation in wild‐type and HDAC6 KO MEF cells. The degradation of [14C]‐valine labelled long‐lived protein was measured in the presence or absence of 3‐methyl adenine (3MA, inhibits the formation of autophagic vacuoles). The average of percentage degradation from three independent experiments is presented. The error bar represents the standard deviation.(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.(E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.(F) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.(A) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells. Different subcellular fractions (75 μg protein) isolated from the wild type (WT) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Hom, homogenate; APG, autophagosomes; APL, autophagolysosomes; Lys, lysosomes.(B) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.(C) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F‐actin was detected by phalloidin (red). The arrows indicated ubiquitin‐positive protein aggregates.(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).

(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.

(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.(E) The mCherry‐GFP‐LC3 plasmid was cotransfected with wild type, 9KQ (acetylation‐mimic), or 9KR (deacetylation‐mimic) cortactin‐expressing plasmids into wild‐type MEFs. Autophagosome-lysosome fusion was analysed as described in Figure 3A.

(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm

(B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm.(C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI.(D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).