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Real time imaging of dynamic secretory events to define the role of protein O-glycosylation in regulated secretion

Thursday, September 17, 2015 — Poster Session III

3:30 p.m. – 5:00 p.m.
FAES Terrace
NIDCR
CELLBIO-22

Authors

  • DT Tran
  • KG Ten Hagen

Abstract

Regulated secretion is a critical process by which cells deliver molecules to the cell surface and extracellular space. As aberrant secretion is the cause of many human diseases, defining the factors involved in secretion is a crucial step towards developing therapeutic targets for these diseases. Drosophila salivary glands are the largest secretory structures of the fly and secrete mucin-type glycoproteins. Therefore, this organ represents a tractable experimental system for studying factors that regulate hormone-induced secretion. Thus, we performed real-time imaging on transgenic flies containing GFP-labeled secretory cargo (Sgs3-GFP), GFP-tagged myosin II (Zipper-GFP), and RFP-tagged actin binding protein (LifeAct-RFP) to visualize the dynamics of regulated secretion. Our imaging revealed apical and luminal expansion of the salivary glands, as Sgs3-GFP is released from secretory granules in response to the hormone ecdysone. Imaging of labeled actin and myosin revealed that actin is recruited to vesicles after fusion pore formation, followed by myosin recruitment. Inhibition of actin polymerization using latrunculin A disrupted secretion, demonstrating an essential role for actin in secretion, similar to mammalian salivary gland secretion. To address the roles of Golgi-based post-translational modifications on secretion, we next examined the effects of loss of mucin-type O-linked glycosylation. RNA interference (RNAi) to genes responsible for initiating this conserved protein modification resulted in irregularly shaped granules with compromised membranes and aberrant secretion. Together, our studies define key steps involved in regulated exocytosis in an intact exocrine organ and present the first system to visualize the effects of O-glycosylation on in vivo secretion in real time.

Category: Cell Biology