2013 Theses Doctoral

HDAC6 activity is required for efficient polarization and intracellular transport of organelles in directionally migrating cells

Salam, Ambar Asghar

Deacetylation of non-histone substrates by histone deacetylase 6 (HDAC6) has been shown to affect chemotactic invasion (e.g., Haggarty et al., 2003; Hubbert et al., 2002; Tran et al., 2007) or wound healing motility (Lafarga et al., 2012) of fibroblasts. Chemotactic invasion and wound-healing motility are both examples of directional migration, a process which requires polarization of cytoskeletal and cellular components to facilitate directed organelle and vesicle transport. This ultimately establishes a persistent leading edge and represses random migration. Given that tubulin is a known substrate of HDAC6 and since microtubules (MTs) are known to play an important role in polarization of directionally migrating cells, we hypothesized that HDAC6 activity regulates important MT dependent aspects of directional cell motility. We were able to test this hypothesis by observing cellular polarization and organelle and vesicle transport in the presence or absence of HDAC6 activity using a wound-healing directional motility model. Experiments were carried out in HDAC6 null and wild type fibroblasts, and results were corroborated by pharmacological inhibition of HDAC6 activity. Loss of HDAC6 activity resulted in hyper-acetylation of tubulin, but not of Hsp90 or cortactin, two other HDAC6 substrates known to be involved in actin-mediated regulation of cell motility. Cells lacking HDAC6 activity showed significantly reduced wound-healing velocity but increased lamellipodial protrusions and random migration. These cells also failed to polarize their array of posttranslationally modified MTs as well as organelles such as their MT-organizing center (MTOC), Golgi apparatus, mitochondria, and vimentin filament array. Localizations of cortical dynein and its MT plus-end binding partner and processivity factor, p150Glued, known to be involved in MTOC reorientation via a MT capture mechanism, were also compromised in the absence of HDAC6 activity. In addition, polarized bi-directional transport of Golgi vesicles in motile cells was decreased. Retrograde and anterograde organelle transport were independently affected: Golgi reclustering during recovery from a Golgi inhibitor and mitochondrial trafficking in spreading cells, two canonical dynein- and kinesin- mediated events, were inhibited. Our results were specific to HDAC6 as re-expression in HDAC6 null cells rescued bi-directional Golgi vesicle transport. Testing the specific contribution of the acetylation state of lysine 40 of á-tubulin however, proved inconclusive as both re-expression of wild type á-tubulin and a non-acetylatable mutant equivalently rescued the Golgi vesicle motility defect. We also report initial data from our investigation into two parallel cell polarization pathways reported to orient a stable, modified MT array towards the direction of migration. Localizations of components of a MT-capture complex involved in stable MT array reorientation, cortical LL5â, and its MT plus-end tracking partner, CLASP2, were inhibited by loss of HDAC6, reminiscent of our results with cortical dynein and p150Glued. Paradoxically, localization of EB1, a MT plus-end binding protein involved in an mDia-mediated MT stabilization pathway was not affected. The localization of CLIP170, another MT plus-end binding factor related to p150Glued and known to be important for the recruitment of CLASPs to MT ends, was also not affected. These data provide novel insight into the role of HDAC6 in fibroblast motility showing that its activity is required for regulating MT-based cellular polarization and dynein- and kinesin-mediated vesicular and organellar transport in directionally migrating cells. Our data also point to possible mechanisms by which deregulation of MT acetylation, by abrogating or inhibiting HDAC6 activity, inhibits directional motility and underscore the importance of this MT subset in establishing cellular polarization. We propose that HDAC6 activity modulates bidirectional transport in motile cells by regulating levels of MTs enriched in Ac subunits in response to external cues.