The role and regulation of an actomyosin complex in membrane remodeling during regulated exocytosis in a live rodent model system

Our lab has developed a subcellular imaging technique based on intravital microscopy (IVM) to study the highly conserved process of regulated exocytosis, as it occurs in live rodents, using the submandibular salivary glands (SSGs) as a model system for exocrine secretion. Previous work, mainly done on rats and commercially available transgenic and wild-type mice, determined that an actomyosin complex, composed of F-actin and nonmuscle myosin II (NMII) isoforms A and B (NMIIA and NMIIB, respectively), forms around single secretory granules that have fused with the apical plasma membrane (APM) following β-adrenergic stimulation of regulated exocytosis. This complex is required to promote the full integration of the granules into the APM, and although the role of F-actin in regulating granule integration was established, the roles and regulation of NMIIA and NMIIB in this process were not determined, and therefore, they are the main topics discussed in this thesis. To this end, a series of tools, which includes 1) NMII floxed mice, 2) transgenic and knock-in mice, 3) transgene delivery and expression by viral and non-viral means, and 4) various pharmacological inhibition approaches, were developed. This thesis work shows that: 1) both NMII isoforms are required for granule integration to occur, with NMIIA regulating the dynamics of the fusion pore and NMIIB stabilizing the granules and providing the necessary force for their gradual integration; 2) both NMII isoforms are recruited onto the granules in an F-actin independent manner and subsequently activated by the phosphorylation of their regulatory light chains by myosin light chain kinase (MLCK); and 3) MLCK is recruited through the GTPase Septin 2, and possibly other members of the septin family (Septins 6, 7 and 9) in an F-actin dependent manner. Live imaging by IVM provided further details on the kinetics and dynamics of actomyosin complex assembly and function, which lead to a mechanistic model of actomyosin activity in membrane remodeling during regulated exocytosis in the SSGs. This work is the first to describe how the actomyosin complex functions in a live rodent during a normal physiological process.Ph.D.Includes bibliographical referencesby Oleg Milber