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Fig. 2. Rho activity controls the level of tubulin acetylation upstream of HDAC6. (A) Inhibition of Rho by 0.5 µM TAT-C3 for 5 hours induced an accumulation of acetylated microtubules in comparison to 0.5 µM TAT-GFP used as a control. (B) One nucleus per osteoclast was microinjected with either RhoA WT-GFP or a constitutively activated form of Rho, RhoAV14-GFP expression vectors. Cells were fixed 6 hours after microinjection and GFP-expressing cells were detected by GFP fluorescence using a confocal microscope. Acetylated tubulin was detected by indirect immunofluorescence (green) and F-actin by means of phalloidin-RITC (red) and a close-up of each condition is presented. In the presence of RhoA-WT, osteoclasts exhibit the typical podosome belt and dense networks of acetylated microtubules. On the other hand, expression of Rho V14-GFP induced deacetylation of microtubules and disorganisation of podosome belts (arrowhead in close-up area). However, tubulin deacetylation dependent on Rho activation was inhibited after treatment with the HDAC6 inhibitor TSA (3 µM) for 1 hour, showing that this enzyme is downstream of Rho. (C,D) HDAC6 is present and active in osteoclasts. Endogenous HDAC6 was easily detected in osteoclasts by western blotting with a polyclonal anti-HDAC6 antibody (C). The deacetylase activity of HDAC6 was tested with two drugs: TSA, known to inhibit its activity and sodium butyrate, which does not. HDAC6 was indeed active in osteoclasts as confirmed by greatly increased levels of acetylated tubulin in TSA-treated osteoclasts and unchanged levels in the presence of sodium butyrate compared to the control and to the total amount of ß-tubulin (D). Finally, inhibition of Rho by TAT-C3 (for 4 hours) in the presence of TSA had no additional effect on the increase in acetylated tubulin (C). Bar, 20 µm.
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