Role of clathrin-coated plaques in regulating cell migration

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Clathrin is a unique scaffold protein, which forms polyhedral cages at the plasma membrane assembling into flat and curved lattices. The function of the curved clathrin-coated pits in forming endocytic structures is well studied. On the contrary, the role of the flat hexagonal clathrin arrays, also known as plaques remains ambiguous. Several studies have suggested that plaques are involved in cell adhesion however, the molecular origins and mechanisms leading to the formation of such flat clathrin lattices are unknown. Furthermore, the physiological relevance and functions of plaques remains to be determined. In preparation of this proposal, we identified the mechanisms leading to plaque formation and their functions during cell migration. In migrating cells, focal adhesions at the leading edge disassemble in a co-ordinated manner. Using live-cell microscopy, we could demonstrate that these focal adhesions get replaced by clathrin-coated plaques, a process we termed “focal-adhesion switch”. We could demonstrate that plaque recruitment directly follows after the active digestion of the extracellular matrix by focal adhesions. Using artificial 3D-micropatterns or genetic manipulation of the proteolytic activity of focal adhesions we could show that plaques are recruited, in an integrin-dependent manner, to 3D-topographical cues created by focal adhesions. Through genetic silencing, we have identified Eps15/R as an important regulator of plaque formation during focal-adhesion switch. By performing migration assays of wild type cells and cells depleted of Eps15/R, we could correlate cell directionality and velocity with the ability of plaques to read extracellular topographical cues. As such, we have identified the origin and function of plaques during cell migration and we propose that clathrin-coated plaques may act as regulator of unconventional collective migration in a cell-to-cell contact independent manner. This proposal builds on these preliminary findings and aims at shedding lights on the following opened questions: what are the molecular/biophysical signals that drive clathrin recruitment during focal-adhesion switch and cell migration? What are the molecular components that stabilize a clathrin coat into a clathrin-coated plaque? How do plaques regulate cell migration? The specific aims will be:Aim 1: Defining the importance of integrins and topographical cues in regulating plaque formation and stability Aim 2: Function of Eps15/R, from mediating FA switch to regulating cell migrationAim 3: Identification of the molecular determinants regulating plaque formation and stability