Bazooka and atypical Protein Kinase C regulate dynamic actomyosin networks during Drosophila amnioserosa apical constriction

Cell shape changes drive tissue remodeling. Polarized activity of actin and myosin drive apical constriction, as seen for example in Drosophila embryonic amnioserosa cells during dorsal closure (DC). My first aim was to characterize interactions between apical actomyosin networks and the cell polarity regulators Bazooka (Baz, the Drosophila Par-3), Par-6, and atypical Protein Kinase C (aPKC) (the PAR complex) during Drosophila DC. I found that both actomyosin networks and the PAR complex are enriched at the apical surfaces of amnioserosa cells and that actomyosin contractility is driven by cyclical assembly and disassembly of actomyosin networks. The pulsatile actomyosin networks translocate across persistent apical surface PAR complex puncta. To assess whether the PAR complex interacts with actomyosin, I characterized myosin dynamics with PAR loss- and gain-of-function perturbations. Baz enhances, whereas Par6/aPKC inhibits actomyosin. These studies suggest that PAR proteins regulate pulsatile apical actomyosin networks mediating constriction of amnioserosa cells. My next aim was to characterize actomyosin networks during a shift in their dynamics. Amnioserosa constriction transitions from pulsatile to persistent from early to late DC. Since oscillatory networks result from delayed negative feedback, I examined whether such regulation exists in these actomyosin networks. The actomyosin inhibitor aPKC is recruited to the apical surface by actomyosin and, in turn, aPKC recruits Baz. To examine the significance of Baz – aPKC dynamic interactions, I ectopically stabilized their interactions. This served to inhibit the antagonism of actomyosin networks by aPKC, suggesting that Baz can act as a competitive inhibitor of aPKC. I found that interactions between Par-6 and Baz increase during DC, suggesting an increase in aPKC inhibition during DC progression. To examine whether decreased inhibition can tune oscillatory actomyosin networks, we collaborated with Qiming Wang and Dr. James Feng (University of British Columbia) to test this oscillation in silico. Computer modeling suggests that decreasing delayed negative feedback can transition actomyosin oscillations towards stabilized constriction. Together, these results demonstrate the requirement of Baz – aPKC interactions for their localization and for dynamic regulation of aPKC by a competitive inhibitor. Finally, my research reveals a regulatory circuit to tune actomyosin behaviour during development.Ph