Visualization of exocytosis and vesicle trafficking in inner medullary collecting duct (IMCD)

Abstract

Conventional confocal microscopy has been used to monitor vasopressin (AVP) induced apical exocytosis in perfused IMCD using FM1‐43 as a membrane tracer, but individual fusion event could not be resolved. In two‐photon confocal microscopy, two‐photon excitation does not excite tracer molecules outside of the focal plane, and it can simultaneously excite multiple tracers with a single laser source. We sough to determine whether two‐photon confocal microscopy can detect individual vesicle fusion event in IMCD using a membrane tracer (FM1‐43) and a fluid‐phase tracer (Sulforhodamine B, SRB) simultaneously. Luminal FM1‐43 and SRB were excited at 850 nm. AVP (1 nM) induced a gradual increase of FM1‐43 fluorescence in the apical membrane, indicating an increase of membrane surface area due to exocytosis. Opening and flattening of fusion pores were also observed in the apical membrane. At the site of membrane fusion, FM1‐43 emission was increased ahead of SRB emission, which suggested that FM1‐43 diffuses along the lipid membrane into the vesicle while SRB enters the vesicle after the fusion pore is opened. The SRB emission dropped after the vesicle was flattened. The size of vesicles varied between 0.5 to 1.5 μm. The fusion time ranged from 30–80 s. Transcellular trafficking of fluorescent vesicles was also observed. These results demonstrated the feasibility to monitor vesicle fusion and trafficking in perfused IMCD.