Determinanti della polarità cellulare: ruolo degli stimoli polarizzanti extracellulari nell'attivazione e mantenimento di programmi genetici e funzionali in organismi multicellulari

The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known.

Myosin VI was originally discovered in Drosophila, where it was shown to participate in cell migration during ovary development, in spermatogenesis and in neuroblasts asymmetric divisions. Myosin VI moves toward the pointed end of actin filaments, directionality distinct from all other characterized members of the myosin family, which move towards the barbed end. Current evidence suggests that it has roles both in particle transport and in pulling membranes down along polarized actin filaments. An attractive hypothesis is that myosin VI associates with cell adhesion molecules at the leading edge, and by moving towards the pointed-end of actin tracts, it contributes to membrane protrusions and ultimately to the propulsion of the cell body. Accordingly, in mammalian cells, myosin VI has been localized at the Golgi complex, in membrane ruffles at the leading edge, and in clathrin coated pits at the plasma membrane. It is conceivable that the link between the endocytic compartment and cellular proliferation and migration is through regulation of epithelial polarity. More research is needed to determine whether these compartments are connected through myosin VI or whether myosin VI assumes separate roles in regulating endocytosis, cell polarity, and cell migration.

In spite of the increasing interest, little is to date known about the molecular mechanisms regulating myosin VI´s action. Preliminary results from our lab revealed the presence of two ubiquitin binding domains (UBDs) in the C-terminal portion of myosin VI, making a strong case for involvement of Ub-signaling in the control system of the myosin VI motor functions. Following these observations, in this proposal we aim at addressing the functional role and the potential implications in cell migration for the association of ubiquitinated-proteins to myosin VI. Our experimental plan is designed to deliver an accurate biochemical and high-resolution structural description of myosin VI UBDs, and of their ubiquitinated interactors. This information will be fueled into cell biology assays to test the relevance of Ub-association to myosin VI in living cells. In line with the current knowledge, our working hypothesis is that Ub-binding may assist dimerization of the myosin VI tail, and hence contribute to cell migration/cell polarity by enhancing the processive transport of cargo molecules along actin filaments. Alternatively, the UBDs of myosin VI may serve as docking sites for the recruitment of ubiquitinated cargo molecules under the control of a more intricate Ub-signaling network. To test these ideas, we will characterize the organizational principles of myosin VI:Ub complexes and assess the migrating phenotypes of cells in which endogenous myosin VI has been replaced with mutants impaired in Ub-binding. We also expect to obtain valuable insights into the molecular pathways controlling myosin VI by the characterization of its ubiquitinated cellular interactors, which have been already identified by mass spectrometry-based methodologies. In summary, we plan to use a multidisciplinary approach involving structural and cellular biology to uncover the molecular mechanisms accounting for the role of myosin VI during endocytosis, cell polarity, and cell migration.