Abstract
Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.
Highlights
Cellular secretion via regulated exocytosis is pivotally influenced by cell cytoskeleton
Secretory vesicles in alveolar type II (ATII) cells are surrounded by microtubule network
To investigate whether microtubules influence actin coat formation and function in ATII cells we first explored the spatial relationship between the microtubule network and secretory vesicles using immunostaining and electron microscopy
Summary
Cellular secretion via regulated exocytosis is pivotally influenced by cell cytoskeleton. Actin and microtubule crosslinking proteins were suggested to connect both cytoskeletal networks and act as mediators in signalling cascades to control cytoskeletal remodelling[23]. One such protein is IQGAP1, which binds to actin directly[43] and to microtubules indirectly via CLIP17044. Experimental evidence suggests that actin and microtubules can interact with each other, the nature and significance of this interaction during exocytosis is not clear We addressed this question in surfactant-secreting primary alveolar type II (ATII) cells. In ATII cells, vesicle content extrusion is facilitated by actin coat contractility, which is visible as shrinkage of actin ring in epifluorescence microscopy[12,13,54].
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