Investigating the molecular mechanism of Shroom-Rock interaction and its role in cellular and tissue morphogenesis

During development cells undergo highly synchronized, dynamic morphogenetic movements to pattern the complex 3D architecture of tissues and organs involving processes like cell adhesion, migration, shape and polarity. This cellular remodeling is brought about by proteins that can spatially and temporally modulate the dynamics and organization of the cytoskeleton. Shroom is a class of actin-associated proteins that has been implicated in regulating cell and tissue architecture. Shroom3 along with another cytoskeleton regulator protein Rock, a kinase, locally activates non-muscle myosin II and facilitates assembly of a contractile actomyosin network at the apical surface of cells which subsequently alters cell shape and behavior. In order to elucidate the molecular mechanism and dynamics of Shroom-Rock interaction it becomes important to map the residues mediating this interaction. My project aims to dissect the molecular nature of Shroom-Rock interaction using a variety of biochemical and cell based assays, guided by structural studies and novel Shroom3 mutants. Shroom SD2 is known to form a three segmented antiparallel coiled-coil dimer. We have identified a highly conserved patch of surface exposed residues in Shroom SD2 that are important for Rock binding and interiorly buried residues required for Shroom dimerization. I have shown that the SD2 mutants that fail to bind Rock or dimerize also fail to cause apical constriction in MDCK cells. I have also shown specifically an Arginine residue in Shroom SD2 to be essential for Rock binding, apical constriction and neural tube morphogenesis in mice. Next, we mapped the SBD of Rock to a stretch of 79 amino acids in its coiled--coil region which is highly conserved across species. Using mutational analysis as well as in vitro and in vivo assays I have identified surface patches of highly conserved residues in Rock SBD that are important for Shroom binding, co-localization with Shroom, and apical constriction of MDCK cells. These results supplemented by the crystal structure of Rock SBD have facilitated a better understanding of the dynamics of Shroom-Rock interaction and ultimately cell morphogenesis. Overall, elucidating the Shroom-Rock interaction has helped establish an evolutionarily conserved signaling module as a paradigm for cellular and tissue morphogenesis.