Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca 2+ concentration ([Ca 2+ ]) c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca 2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca 2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca 2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca 2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X 4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X 4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X 4 . Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca 2+ influx via vesicular Ca 2+ channels regulates fusion pore expansion and vesicle content release.egulated secretion is a fundamental cellular process in many different types of eukaryotic cells, with Ca 2+ -triggered exocytosis being the key element (1-4). Multiple Ca 2+ -dependent steps have been elucidated that ultimately lead to fusion of exocytic vesicles with the plasma membrane, resulting in formation of an aqueous channel, the fusion pore, through which vesicle contents are released (5-8). Although the molecular composition of the fusion pore remains elusive, there is a general acceptance that fusion pores are not merely passive structures, but that their opening and closure are highly regulated and control, or even fine-tune, vesicle content secretion (9-14). Voltage-gated Ca 2+ channels are not present (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).We recently reported a "fusion-activated" Ca 2+ entry (FACE) mechanism as a phenomenon in the postfusion phase of surfactant secretion (29). Given that this Ca 2+ signal occasionally spreads throughout the cell, we speculated that it might be important for triggering...

SummarySecretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required.

Type II pneumocytes secrete surfactant, a lipoprotein-like substance reducing the surface tension in the lung, by regulated exocytosis of secretory vesicles termed lamellar bodies (LBs). This secretory process is characterized by a protracted postfusion phase in which fusion pores open slowly and may act as mechanical barriers for release. Combining dark-field with fluorescence microscopy, we show in ss-actin green fluorescent protein-transfected pneumocytes that LB fusion with the plasma membrane is followed by actin coating of the fused LB. This is inhibited by cytoplasmic Ca(2+) chelation or the phospholipase D inhibitor C2 ceramide. Actin coating occurs by polymerization of actin monomers, as evidenced by staining with Alexa 568 phalloidin. After actin coating of the fused LB, it either shrinks while releasing surfactant ("kiss-coat-and-release"), remains in this fused state without further action ("kiss-coat-and-wait"), or is retrieved and pushed forward in the cell on top of an actin tail ("kiss-coat-and-run"). In the absence of actin coating, no release or run was observed. These data suggest that actin coating creates a force needed for either extrusion of vesicle contents or retrieval and intracellular propulsion.

In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion.

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