Abstract
The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo.
Highlights
The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion
This stimulus induces the expression of the sgs genes that encode the cargo that will be packaged into secretory vesicles; a second pulse of 20E during late third instar instructs these newly formed secretory granules to fuse with the apical plasma membrane (PM) to begin secretion[31,32,33]
The diameter of the lumen expands as it fills with the Sgs3-GFP cargo (Fig. 1b and Supplementary Movie 1), similar to what is seen in vivo (Supplementary Fig. 1a). (Real-time videos of all still images shown throughout this paper are in the Supplementary Movies section.) Imaging the dynamics of individual secretory granules using Drosophila lines expressing Sgs3-GFP cargo and the apical PM marker Myr-tdTomato revealed apical membrane deformations at the point of vesicle fusion (Fig. 1c)
Summary
The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. We show essential roles for the branched actin nucleators Arp2/3and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Regulated exocytosis is a fundamental cellular process that involves the release of stored secretory cargo from membranous vesicles into the extracellular space. This type of secretion involves the formation of secretory vesicles and their subsequent docking and fusion with the plasma membrane (PM) in response to an external stimulus. The role of branched actin assembly and structure is less well understood in other forms of secretion that involve large secretory vesicles containing bulky cargo[3]. Limited genetic tools in many systems have restricted the ability to interrogate the function of specific genes involved in actin formation in vivo
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