Figure 7.

Enhanced tubulin deacetylation caused by GRK2-mediated HDAC6 phosphorylation modulates cell spreading kinetics and motility. (A) Impairment of GRK2-mediated HDAC6 phosphorylation or pharmacological inhibition of HDAC6 accelerates spreading. The spreading kinetics of parental and HeLa-wt5 pretreated or not with TSA (1 mM) or HeLa-A1 or -K1 cells was analysed using the XCELLigence system as detailed in Materials and methods. (B) Gene-targeted inactivation of GRK2 increases the rate of fibroblast spreading. Primary MEFs derived from GRK2-floxed mice were infected with control or Cre-recombinase expressing adenovirus and their spreading was analysed as above. Total time needed to achieve a maximum cell index during spreading and the extent of tubulin acetylation at this stage was determined for each cell line. Data are mean±s.e.m. of three independent experiments. ^*P<0.05, ^**P<0.01, compared with HeLa parental cells or control infected MEFs, unpaired two-tailed t-test. (C) Both the extent and time course of tubulin acetylation during cellular spreading are altered in the presence of GRK2 mutants defective in HDAC6 phosphorylation. Parental and HeLa-wt5, -A1 and -K1 cells were kept in suspension for 2 h and then allowed to adhere and spread into FN-coated plates for the indicated times. Acetylated α-tubulin and total α-tubulin levels were immunodetected with specific antibodies. A representative blot and quantification of tubulin acetylation are shown. (D) Levels of GRK2-pS670 are differentially regulated during spreading and inversely correlate with the spreading rate. Cells were serum starved and collected after kept in suspension (S) or allowed to adhere and spread for 1 h (A) onto FN-coated plates. The extent of GRK2 phosphorylation at S670 and total GRK2 levels were analysed by western blot. A representative blot from two independent experiments is shown. (E) HDAC6-induced migration requires phosphorylation of C-terminal residues on HDCA6 by GRK2. HeLa cells were co-transfected with the CD-8 antigen in the presence of HDAC6-wt or HDAC6-S1060,1062A or S1060,1062,1069A mutants and sorted using microbeads precoated with anti-CD8 antibody. Cell migration was assessed as detailed in Materials and methods. ^*P<0.05, compared with HDAC6-wt transfected cells (unpaired two-tailed t-test). (F) Expression of a HDAC6 mutant defective in GRK2 phosphorylation accelerates cell spreading kinetics. HeLa cells transfected with GFP-HDAC6-wt or mutant GFP-HDAC6-S1060,1062,1069A were plated on coverslips coated with FN (10 μg/ml), fixed at the indicated times and analysed by confocal microscopy. The spreading area of GFP positive (green labelled) and negative cells was quantified by morphometric analysis and cells were double stained for acetylated α-Tubulin (blue) and F-actin (Phalloidin, red) as in Figure 6. Plotted data are mean±s.e.m. from 10 to 30 cells for each time point and cellular condition. Figure source data can be found in Supplementary data.