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Fig. 3. mDIA2 mediates the action of Rho at the level of acetylated microtubules by interacting with and activating HDAC6. (A) Constitutively active mDia2 blocks tubulin acetylation and podosome belt formation. One nucleus per osteoclast was microinjected with either GFP or a constitutively activated form of mDIA2, GFP-mDIA2 
GBD expression vectors. 6 hours later, acetylated tubulin was detected by indirect immunofluorescence (green) and F-actin with phalloidin-RITC (red). In GFP-mDIA2 GBD-expressing osteoclasts compared to GFP-expressing osteoclasts, the Ac-MT level was dramatically decreased and the podosome belt disrupted. (B) HDAC6 and mDia2 interact together. COS cells were either transfected with GFP alone, HA-HDAC6, GFP-mDIA2 WT, GFP-mDIA2 GBD vectors or co-transfected together with HDAC6. Transfected HDAC6 or mDia2 were revealed in total cell lysate (TCL) by an anti-HA or an anti-GFP antibody respectively (left panel). HDAC6 was immunoprecipitated with an anti-HA antibody and mDia2-associated proteins were revealed with an anti-GFP antibody (right panel). Both the wild type and activated mDia2 co-precipitated with HA-tagged HDAC6. (C) Mapping of HDAC6 domain interaction with mDia2. To determine the domains of HDAC6 implicated in the interaction with GFP-mDIA2 WT, COS cells were transfected with GFP-mDIA2 WT and with the N-terminal domain, the deacetylase domain 1 (DD1), the deacetylase domain 2 (DD2) or the C-terminal domain of HDAC6, using HA-tagged expression vectors. Cell lysates of transfected cells were immunoprecipitated with a monoclonal anti-HA antibody. (D) mDia2 increases HDAC6 deacetylase activity. In a tubulin deacetylase in vitro assay, COS cells were transfected with mHDAC6-HA WT, lysed at room temperature for 15 minutes and the ratio of acetylated tubulin/tubulin monitored by western blotting. When GFP-mDIA2 WT was transfected with mHDAC6-HA WT, this increased the deacetylase activity of the enzyme whereas GFP-mDIA2 WT alone had no effect on the level of this PTM. Positions of protein standards in kDa are indicated on the left-hand side of blots.
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