Journal: The Journal of Cell Biology
Article Title: Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition
doi: 10.1083/jcb.201702187
Figure Lengend Snippet: Miro1 K105Q is an axonal substrate for HDAC6 after exposure to CNS growth inhibitors. (A) End-point FRAP for Mito-GFP recovery in distal axons of DRGs transfected with WT, acetyl-mimetic (K40Q), or nonacetylatable (K40A) α-tubulin constructs and plated onto laminin is shown as average of normalized percentage recovery ± SEM at 960 s after bleach. Exposure to 10 µM TubA significantly increases Mito-GFP recovery in all three conditions (*, P ≤ 0.05 for TubA-treated vs. its corresponding DMSO control by two-way ANOVA with Tukey post hoc). Fig. S6 (A and B) shows that these mutant α-tubulin proteins are incorporated into axonal microtubules and expressed at relatively equivalent levels. (B) Representative immunoblot is shown for Miro1 from input and immunoprecipitations with magnetic bead-conjugated nonimmune IgGs or anti–Ac-Lys antibody cocktail from DRG neurons treated with 10 µM TubA for 4 h. 10% of the protein lysate was used as input (pull-down efficiency of 10.1 ± 1.9% for control vs. 14.1 ± 2.2% for TubA over n = 4; P = 0.012 by two-tailed Student’s t test). (C) HDAC6 is detected in Miro1 immunoprecipitates, and Miro1 is detected in HDAC6 immunoprecipitates from cultured DRG neurons by immunoblotting. 10% of the protein lysate was used as input. (D) Schematic of rat Miro1 sequence with residues previously reported to be acetylated in nonneuronal cells indicated (K105, K525, and K629 plus (Ac)). The glutamate-to-lysine mutations that were previously reported to decrease Miro1 Ca 2+ sensitivity are indicated by (KK). Residues corresponding to GTPase, EF-hand, and transmembrane (TM) domains are shown. (E) Representative immunoblots are shown for anti-Myc from DRG neurons transfected with Myc-Miro1 K105A , Myc-Miro1 K525A , or Myc-Miro1 K629A plasmids and treated with 10 µM TubA for 4 h at 36 h after transfection (40 h in vitro). Input (10%) and immunoprecipitations with nonimmune IgG and Ac-Lys antibody cocktail. (F) Representative immunoblots are shown for anti–Miro1-AcK105 for DRG lysates ± 10 µM TubA. For the righthand blot, the anti–Miro1-AcK105 antibody was preincubated with 100 µg/ml immunizing peptide (short exposure = 30 s; long exposure = 3 min). (G and H) Representative immunoblots are shown in G for anti–Miro1-AcK105, anti-Miro1, and anti-Erk1 (loading control) from lysates of DRG cultures treated with 10 µg/ml aggrecan (CSPG) or 1 µM thapsigargin (Thapsi) for 4 h. H shows quantification of immunoblot signals across multiple experiments as average fold-change relative to control ± SEM ( n = 3; *, P ≤ 0.05 by one-way ANOVA with pairwise comparison and Tukey post hoc tests). (I and J) Representative confocal projection images (XYZ) for anti-Miro1-AcK105 (Cy5), anti-NF (Cy3), and MitoTracker Green are shown as indicated in J for control, aggrecan-treated (CSPG) or thapsigargin-treated cultures. ImageJ was used for pseudocoloring and channel merging. Panel I shows quantification of the axonal anti-Miro1-AcK105 signals under these conditions as average fold-change relative to control ± SEM ( n = 20; *, P ≤ 0.05 by one-way ANOVA with pairwise comparison and Tukey post hoc; scale bar = 20 µm; 100×/1.4 NA objective used).
Article Snippet: Cells were washed three times in PBST and then incubated with Cy5-conjugated anti-mouse IgG (1:200; Jackson Immunoresearch) in 1% blocking buffer for 1 h. Cells were washed twice with PBST and then incubated with the Alexa Fluor 405–conjugated mouse 6-11-B1 anti–Ac-α-tubulin (1:50; Sigma-Aldrich; T6793) and FITC-conjugated Tub 2.1 mouse anti–β-tubulin IgG (1:50; Sigma-Aldrich; T4026) in 1% blocking buffer for 1.5 h. Coverslips were washed twice with PBS then stained with Actin-Red 555 Ready Probe for 30 min (1:25 dilution of 200 U/ml stock; Thermo Fisher Scientific; R37112).
Techniques: Transfection, Construct, Mutagenesis, Western Blot, Two Tailed Test, Cell Culture, Sequencing, In Vitro
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