SCA14 mutant γPKC preferably binds with Hsc70 and suppresses CMA in primary cultured PCs.

<p>(<i>A</i>) Schematic illustration of pull-down assay using the HT system. Resin-conjugated HT ligands covalently bind to γPKC-HT and harvest γPKC-HT and γPKC-binding proteins from cell lysate. After washes and heat treatment, γPKC-binding proteins are released from the resin, but γPKC-HT itself is still resin-bound through the covalent linkage between HT and the HT ligand. This is a unique feature of the HT pull-down assay, in contrast to other pull-down assays using glutathione S-transferase or immunoprecipitation, and enables us to effectively analyze binding partners of the target protein. (<i>B</i>) Representative silver stained gel of pulled down proteins by the HT pull-down assay and subjected to SDS-PAGE. The HT pull-down assay was conducted using cell lysate from cerebellar primary cultures that expressed WT or mutant (S119P and G128D) γPKC-HT in PCs. The arrow indicates the protein band that was more strongly detected in pull-down samples from cell lysates expressing mutant γPKC-HT than in the WT. Mass spectrometric analysis revealed that this protein is Hsc70. Silver staining gel with pull-down, input and supernatant (Sup) samples is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031232#pone.0031232.s006" target="_blank">Figure S5A</a>. (<i>C</i>) Representative fluorescent images of γPKC-GFP (upper panels) and GAPDH-HT (lower panels) in PC somata 21 h after labeling with TMR-HT ligand. PCs coexpressed GAPDH-HT and either WT (left), S119P (center) or G128D (right) mutant γPKC-GFP. We evaluated GAPDH-HT lysosomal translocation in PCs not displaying aggregation of mutant γPKC-GFP. Bar = 5 µm. (<i>D</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in PC somata in the absence (open bars) or presence (hatched bars) of 100 µM H₂O₂. Mutant versions of γPKC-GFP (S119P and G128D) significantly decreased the number of GAPDH-HT dots (* p<0.05, ** p<0.001 vs PCs expressing WT γPKC-GFP without H₂O₂, unpaired <i>t</i>-test, n = 45, and § p<0.001 vs PCs expressing WT γPKC-GFP with H₂O₂, unpaired <i>t</i>-test, n = 45). While H₂O₂ significantly increased the number of GAPDH-HT dots in PCs expressing WT γPKC-GFP († p<0.01, unpaired <i>t</i>-test), it failed to increase the number of GAPDH-HT dots in PCs expressing mutant γPKC-GFP (n.s.: not significant (p>0.05), unpaired <i>t</i>-test).</p

Lysosomal translocation of GAPDH-HT in primary cultured PCs.

<p>(<i>A</i>) Representative fluorescent images of TMR-labeled GAPDH-HT immediately after (0 h, left) and 24 h after labeling (24 h, right) with TMR-HT ligand in primary cultured PCs. Images were projected from a Z-stack of images obtained by confocal laser microscopy. Bar = 20 µm. (<i>B</i>) Representative GAPDH-HT fluorescence (left), LAMP2 immunostaining (center) and merged (right) images of PC somata 21 h after labeling with TMR-HT ligand. Images were taken from the center of the Z-stack. Bar = 5 µm. (<i>C</i>) Representative GAPDH-HT fluorescence (left), LysoTracker red fluorescence (center) and merged (right) images of PC somata 21 h after labeling with TMR-OG ligand. Bar = 5 µm. (<i>D</i>) Representative fluorescent images of GAPDH-HT in PC somata treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), H₂O₂ (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower) and 6-aminonicotinamide (6-AN; 1 mM, right lower). Bar = 5 µm. (<i>E</i>) Quantitative analyses of lysosomal translocation of GAPDH-HT in PC somata treated with CMA activators. Dots of GAPDH-HT in each PC soma were counted in the center image from the Z-stack. Numbers of GAPDH-HT dots were significantly increased in the presence of CMA activators (H₂O₂, MPA and 6-AN). * p<0.01 vs PCs treated with vehicle (unpaired <i>t</i>-test, n = 60 for cells treated with vehicle, n = 30 for cells treated with CMA activators).</p

Lysosomal translocation of GAPDH-HT reflects CMA activity in HeLa cells.

<p>(<i>A</i>) Representative fluorescent images of GAPDH-HT 21 h after labeling with TMR-HT ligand in HeLa cells transfected with nontargeting-siRNA (left) or LAMP2A-siRNA (right). Bar = 20 µm. (<i>B,C</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells transfected with nontargeting (Non)- and LAMP2A-siRNA. (<i>B</i>) Cells having more than 5 dots of GAPDH-HT were classified as GAPDH-HT dot-positive cells. We counted the number of GAPDH-HT dot-positive cells in 50–70 GAPDH-HT-expressing cells. (<i>C</i>) We assessed the number of GAPDH-HT dots per cell. The percentage of GAPDH-HT dot-positive cells and the number of GADPH-HT dots per cell were significantly decreased by siRNA-mediated LAMP2A-knockdown. ** p<0.001 vs cells treated with nontargeting-siRNA (unpaired <i>t</i>-test, n = 16 in <i>B</i>, n = 57 for nontargeting-siRNA and n = 74 for LAMP2A-siRNA in <i>C</i>). (<i>D</i>) Representative fluorescent images of GAPDH-HT taken 21 h after labeling with the TMR-HT ligand in HeLa cells treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), serum free medium (0.1% DMSO, 0.1% methanol, center upper), H₂O₂ (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower), SB202190 (20 µM, left center) or cycloheximide (CHX; 20 µg/ml, right lower). Bar = 20 µm. (<i>E,F</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells treated with CMA activators or inhibitors the percentages of GAPDH-HT dot-positive cells (<i>E</i>) and the number of GAPDH-HT dots per cell (<i>F</i>). Percentages of GAPDH-HT dot-positive cells and the numbers of GAPDH-HT dots per cell were significantly increased by CMA activators (serum free medium, H₂O₂ and MPA), while they were significantly decreased by CMA inhibitors (SB202190 and CHX). * p<0.01, ** p<0.001 vs cells treated with vehicle (unpaired <i>t</i>-test, n = 12 for cells treated with vehicle, n = 8 for cells treated with CMA activators and inhibitors in <i>E</i>, n = 96 for vehicle, n = 61 for serum free, n = 45 for H₂O₂, n = 58 for MPA, n = 52 for SM202190 and n = 41 for CHX in <i>F</i>).</p

Visualization of CMA substrate translocation from cytosol to lysosomes using the HaloTag (HT) system.

<p>(<i>A</i>) Schematic illustration of CMA substrate-HT labeling with the HT ligand fused to fluorescent dye (gray circle). CMA substrate-HT in cytosol can be labeled with fluorescent dye since the HT reaction occurs at neutral pH. In contrast, CMA substrate-HT in lysosomes cannot be labeled since the HT reaction does not occur at acidic pH. (<i>B</i>) Schematic illustration of CMA substrate-HT translocation from cytosol to lysosome. Because the fluorescent dyes used in this study (TMR and OG) are not quenched at acidic pH, labeled CMA substrates can be visualized after translocation to lysosomes. (<i>C</i>) Representative fluorescent images of TMR-labeled GAPDH-HT immediately after (0 h, upper) and 21 h after labeling (21 h, lower) with TMR-HT ligand in HeLa cells. Arrows on the lower image indicate the cells with obvious GAPDH-HT dots. Bar = 20 µm. (<i>D</i>) Representative GAPDH-HT fluorescence (left), LAMP2 immunostaining (center) and merged (right) images of HeLa cells immediately after (0 h, upper panels) and 21 h after labeling (21 h, lower panels) with TMR-HT ligand. Bar = 5 µm. (<i>E</i>) Representative GAPDH-HT fluorescence (left), LysoTracker red fluorescence (center) and merged (right) images of HeLa cells 21 h after labeling with OG-HT ligand. Bar = 5 µm. (<i>F</i>) Representative GAPDH-HT fluorescence (left), LAMP2A immunostaining (center) and merged (right) images of HeLa cells 21 h after labeling with TMR-HT ligand. Bar = 5 µm.</p