Imaging developmentally‐regulated secretory granule biogenesis and exocytosis

Abstract

Regulated secretion is an essential process by which cells deliver molecules to the cell surface and extracellular space in response to an external signal. We have established a system to directly visualize the secretion of high molecular weight cargo such as mucins in vivo, to define the critical factors at each step in this process. Using Drosophila salivary glands expressing fluorescently‐labeled secretory cargo (Sgs3‐GFP), myosin II (Zipper‐GFP), actin binding protein (Lifeact‐Ruby) or a PIP2 reporter (PLC□PH‐EGFP), we are able to visualize the spatial and temporal dynamics of regulated secretion. Through 4D‐imaging of secreting glands, we show that actin is cleared at the site of vesicle fusion and re‐assembled directionally around secreting granules. Moreover, we demonstrate that fusion pore formation and PIP2 redistribution precede actin and myosin recruitment to fused granules. Additionally, we show a crucial role for the branched actin nucleators Arp2, Arp3 and their nucleation promoting factor WASp in cargo expulsion and integration of the granular membrane with the apical plasma membrane. By combining in vivo and ex vivo imaging of fluorescently‐labeled molecules with the power of Drosophila genetics, we have developed a tractable experimental system for studying the factors that regulate hormone‐induced secretion during development. Ongoing experiments focus on investigating the role of O‐glycosylation in secretory granule biogenesis, morphology and secretion.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIDCR, National Institutes of Health (Z01‐DE‐000713 to K.G.T.H.).