The role of HDAC6 and microtubule acetylation in the regulation of cellular motility

Acetylation is a well-recognized reversible post-translational modification of stabilized microtubules (MTs). However, the cellular functions of MT acetylation are only beginning to be elucidated. Alterations in activity of the MT-selective deacetylase, HDAC6, results in a parallel change in motility and regulates cellular processes, including minor metastasis. Directed migration requires the MT cytoskeleton; stable MTs allow cell polarization, while dynamic MTs target and mediate focal adhesion turnover, permitting continued forward translocation. Whether acetylation, through modulation of MT properties influences MT-dependent cell migration is not known, and in this thesis, we examined the role of HDAC6-mediated MT deacetylation in motility regulation. We also conducted a proteomic screen in order to identify novel HDAC6 cytoskeletal effectors.;We observed that loss of HDAC6 activity, resulting in an increase in MT acetylation, causes an increase in focal adhesion area, corresponding to a decrease in the rate of migration. Live measurements of fluorescently-labeled adhesions revealed that loss of HDAC6 inhibits adhesion turnover, without affecting adhesion assembly. This inhibition in adhesion turnover was specific to the increase in MT acetylation, as expression of non-acetylatable alpha-tubulin is sufficient to rescue adhesion area in HDAC6 KO cells.;We also found that increased MT acetylation dampens MT dynamics: real-time measurements of fluorescently-labeled MTs demonstrated decreased dynamicity and hyperacetylated MTs displayed increased stability against nocodazole-mediated depolymerization. Dynamic MT targeting induces focal adhesion disassembly through the activation of dynamin. We observed that hyperacetylated MTs had a decreased rate of adhesion targeting, which could contribute to the inhibition of adhesion turnover. We have also begun to address possible mechanisms by which increased MT acetylation inhibits adhesion turnover. Overexpression of dynamin2 is able to rescue adhesion area in HDAC6 KO cells, suggesting dynamin activation is impaired upon MT hyperacetylation and we have begun investigating the role of PLD2 as a MT-dependent dynamin stimulator in MT acetylation impaired focal adhesion turnover.;Additionally, we identified a number of proteins both over- and under-represented in the cytoskeletons of HDAC6 KO cells. Over-represented proteins included: lamin A, septin 11, gelsolin, Hsp70 4L, and myosin light chain 6, while under-represented proteins included: alpha-catenin and ezrin. These identified proteins will serve as the basis for future studies into the role of HDAC6 cytoskeletal regulation.