Imaging the Stages of Exocytosis in Epithelial Type II Pneumocytes

Exocytosis proceeds through distinct stages. Based on morphological and functional criteria, they can be classified as pre- and hemifusion, fusion, and postfusion. During the prefusion stage, plasma and vesicle membranes approach each other. During the hemifusion stage, the outer membrane of the vesicle merges with the inner leaflet of the plasma membrane. During the fusion stage, both leaflets of plasma and vesicle membranes are fully merged, and the lumen of the vesicle opens to the extracellular space via an aqueous, small, and pore-like channel. During the postfusion stage, the fusion pore may further expand, and the vesicle content can be entirely expelled. The possibility to capture these events with imaging techniques depends on the speed they occur and the vesicle size in combination with the optical method used (temporal and spatial resolution). Type II pneumocytes of the lung have large (>1 μm) vesicles, termed lamellar bodies (LBs), which are useful for studies of exocytosis with live cell imaging techniques. Fluorescent dyes can be targeted to distinct compartments, and a certain exocytotic stage can be studied by measuring stage-specific diffusion and/or fluorescence enhancement of the selected probes. This paper overviews optical techniques and fluorescent dyes suitable for the investigation of the stages of LB exocytosis.