Balanced Rho activation and inhibition regulates exocytosis by large secretory vesicles

The small GTPase Rho governs actomyosin-based contractility in a variety of cellular settings, by the parallel induction of actin polymerization and of myosin recruitment and activation. Rho function is regulated by GEFs and GAPs, which stimulate and inhibit Rho activity, respectively. We explore the dynamics and molecular design of this fundamental circuitry in the context of “glue” protein secretion from the epithelium of Drosophila salivary glands, which is achieved via uncommonly large exocytic vesicles. Rho-mediated assembly and contraction of an actomyosin coat that forms around these vesicles upon their fusion with the apical cell membrane, is critical for release of their viscous content into the gland lumen. Our observations suggest that basal levels of Rho-GTP, which diffuse from the apical membrane following fusion, are sufficient to generate a nascent coat, which then recruits the activating element RhoGEF2, implying an amplification mechanism for establishing an actomyosin coat sufficiently robust to enable contraction. This process is counteracted by RhoGAP71E, which is also recruited by actin, and whose inhibitory function is also essential for vesicle contraction. Content release from the giant secretory vesicles thus requires a finely timed balance between Rho activation and inhibition. Our current focus is on determining how temporal order is maintained within this circuitry, particularly since actin appears to serve as a shared recruiting element of both activating and inhibiting factors. A corresponding effort aims to elucidate the mechanisms underlying recruitment and contractile activity of myosin, which is distributed in a non-homogeneous, “stripe”-like pattern on the vesicle surface.