Mechanisms of myosin regulation and function during tissue folding

Abstract

Throughout organismal development, precise three-dimensional organization of tissues is required for proper tissue function. These three-dimensional forms are generated by coordinated cell shape changes that induce global tissue shape changes, such as the transformation of an epithelial sheet into a tube. A model for this transformation occurs early in Drosophila development where approximately 1,000 cells on the ventral side of the embryo constrict their apical sides. Apical constriction drives the formation of a furrow that invaginates, forming a tube, and consequently, a new cell layer in the embryo. Constriction of ventral cells is driven by cycles of assembly and disassembly of actin-myosin networks at the cell apex, called pulses. Pulsatile myosin leads to phases of cellular contraction and cell shape stabilization that result in step-wise apical constriction. While many of the key components of the pathway have been identified, how pulsatile myosin is regulated was previously not well understood. The results presented in this thesis identify mechanisms of regulation of these myosin pulses. First, we demonstrated that cycles of phosphorylation and dephosphorylation of the myosin regulatory light chain are required for myosin pulsing and step-wise apical constriction. Uncoupling myosin from its upstream regulators resulted in loss of pulsatile myosin behavior and continuous, instead of incremental, apical constriction. A consequence of persistent, non-pulsatile myosin is a loss of myosin network integrity as the tissue invaginated. Thus, pulsatile myosin requires tight coordination between its activator and inactivator to generate cycles of myosin assembly, coupled to cellular constriction, and myosin disassembly, associated with cell shape stabilization. Second, we demonstrated that myosin motor activity is required for efficient apical constriction and for effective generation of tissue tension. This work defines essential molecular mechanisms that are required for proper cellular constriction and tissue invagination.