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First published online 27 March 2007
doi: 10.1242/jcs.03431

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HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions

¹ Department of Biological Sciences, Columbia University, New York, NY 10027, USA
² Broad Institute and Chemical Biology Program, Harvard University and Massachusetts Institute of Technology, 320 Bent Street, Cambridge, MA 02141, USA
³ Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
⁴ Department of Pathology and Cell Biology, Colleges of Arts and Sciences and Physicians and Surgeons, Columbia University, New York, NY 10027, USA

View larger version (64K): [in this window] [in a new window]   Fig. 1. Inhibition of tubulin deacetylation decreases cell motility in Transwell chemotactic invasion assays of NIH-3T3 cells. (A) Micrographs of typical fields. (B) Quantification of invasion migration. Treatments were as indicated. Standard deviation is shown. Asterisks mark conditions statistically different from control (P<0.05).

View larger version (60K): [in this window] [in a new window]   Fig. 2. Altered level or activity of HDAC6 alters focal adhesion area. (A) Paxillin immunostaining of typical TC-7 cells, following 2-hour treatments as indicated. Bar, 20 µm. (B) Quantification of adhesion area in TC-7 cells (shown in A) treated with TSA, taxol and Nz (nocodazole), and NaB; see Materials and Methods for details of measurements of paxillin immunofluorescence. (C,D) Time course of the increase in (C) total focal adhesion area, and (D) average area of each individual focal adhesion. (E) Quantification of total focal adhesion area in NIH-3T3 cells stably expressing neomycin plasmid alone (NEO) or WT HDAC6. (F) Quantification of total focal adhesion area in WT, HDAC6 KO and KO MEFs transiently transfected with HDAC6 (HDAC6 rescue MEFs). (G) Quantification of effects of tubacin on total focal adhesion area in TC-7 cells. *, conditions statistically different from controls (P<0.05); ** in B, statistically significant difference both from control and from cells treated with TSA, taxol and Nz. ** in F, statistically significant difference both from WT control and from HDAC6 KO MEFs.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Cell spreading is altered by HDAC6 level. (A) Early events in spreading of HDAC6 WT and HDAC6 KO MEFs, measured using computer-assisted TIRF microscopy to track the boundaries of the cell during spreading on fibronectin-coated coverslips. Top two panels show quantification of spread cell areas at each time interval. Bottom panel shows quantification of the percentage of membrane protrusions (red) and retractions (blue) in the two cell types, calculated as active length along the periphery vs total length along the periphery. The percentage of edge activity (protrusion in red, retraction in blue) was determined at each time by the quotient (active length of the cell periphery) ÷ (total length of the periphery). The warmth of color (green to blue; yellow to red) indicates greater velocity of retractions and protrusions, respectively. Note that the time course selected by the automated analysis software is slightly different for the two cell types in the lower graphs. (B) Long-term spreading of HDAC6 WT and HDAC6 KO MEFs that had been allowed to spread on uncoated-glass coverslips was quantified from the surface area drawn to include all adhesions.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Focal adhesion turnover and disassembly is affected by MT acetylation. (A) Focal adhesion turnover was calculated from FRAP images of WT and HDAC6 KO MEFs expressing paxillin-GFP. Bars, 20 µm. (B) Average half-time (t1/2) of FRAP of adhesions in HDAC6 WT and HDAC6 KO MEFs expressing GFP-paxillin; n>8 adhesions in each type of MEF. (C,D) Rates of adhesion disassembly (C) and assembly (D) were quantified from the pixel-intensity of individual adhesions in time-lapse images of both HDAC6 WT and HDAC6 KO MEFs that were expressing GFP-paxillin; n>12 adhesions in each type of MEF. *, conditions statistically different from control (P<0.05).

View larger version (43K): [in this window] [in a new window]   Fig. 5. MT acetylation dampens MT dynamics. (A) Typical micrographs used to measure MT dynamics in 3x GFP-EMTB-TC-7 cells by MT end-tracking; an edge of a cell is shown with elapsed time in minutes:seconds, and four sample MT ends are shown (a-d). Bar, 10 µm. (B-C) Rates of MT (B) polymerization, (C) depolymerization, and (D) dynamicity, following 30-minute treatment with TSA or taxol, as indicated (see Materials and Methods for details). (E) The percentage of time MTs spend in a stationary phase, i.e. pausing rather than growing or shrinking, in WT, HDAC6 KO MEFs, and KO MEFs transiently transfected with HDAC6 (HDAC6 rescue MEFs). (F) Nocodazole-resistance of MT arrays in HDAC6 WT and HDAC6 KO MEFs. Error bars represent + s.e.m.; *, conditions statistically different from control (P<0.05); **, statistically significant difference both from WT control and from HDAC6 KO MEFs.