FIG. 3.

HDAC6 translocates to actin membrane ruffles and is required for efficient ruffle formation. (A) Wild-type (WT; top panel) or HDAC6 KO MEFs that stably express human HDAC6-FLAG, HDAC6-mut-FLAG, or HDAC6-ΔBUZ-FLAG were stimulated with PDGF-BB (50 ng/ml for 8 min) and stained with phalloidin-Alexa Fluor 488 for F-actin (green) or with an antibody for HDAC6 or FLAG. Endogenous HDAC6 (red, anti-mHDAC6) and FLAG-tagged HDAC6 (red, anti-FLAG) are both detected at dorsal (filled arrows) and peripheral (filled arrowheads, second panel from top) membrane ruffles. Open arrows indicate stress fibers (top panel). Insert in top panel: an enlarged image shows the concentration of HDAC6 at F-actin-containing spots (open arrowheads). Bar = 10 μm. (B) Quantification of PDGF-BB-induced dorsal ruffle formation in various cell types. The graph represents cells with dorsal circular ruffles. Approximately 500 cells were examined from each group. Results are averages plus standard errors of the means. n = 5 for WT and KO cells; n = 4 for reconstituted cells. **, t < 0.01 in two-tailed t test. (C) Wild-type MEFs that stably express GFP control, HDAC6-GFP, HDAC6-mut-GFP, or HDAC6-ΔBUZ-GFP were examined for dorsal ruffle formation as for panel B. Each bar represents the average value plus standard error of the mean from four independent experiments. **, t < 0.01 in a two-tailed and paired t test. (D) Cell lines described for panel C were tested in Boyden chamber migration assays, and relative motility values are graphed. Each bar represents the average value plus standard deviation from three independent experiments with duplicate wells. *, t < 0.05 in a two-tailed and paired t test. (E) 293T cells were cotransfected with HDAC6-ΔBUZ-FLAG and HDAC6-GFP and processed for immunoprecipitation using anti-FLAG, anti-GFP, or control mouse antibody. The precipitates were analyzed by immunoblotting using anti-GFP (left panel) or anti-FLAG (right panel) antibody to show mutual association of HDAC6-ΔBUZ and the full-length HDAC6.