Optical measurements of spontaneous single granule fusion in lactotrophs

Nina Vardjan, Matjaž Stenovec, Marko Kreft, Robert Zorec

Celica Biomedical Sciences Center, Ljubljana, Slovenia; Medical Faculty, University of Ljubljana, Ljubljana, Slovenia

Recently it has been shown that discharge of granule content is slower in resting (~3min) compared to stimulated lactotrophs (several seconds). To further investigate spontaneous granule fusions we used confocal imaging of lactotrophs expressing synaptopHluorin (spH), a granule membrane-targeted pH sensor protein that enables optical discrimination between unfused (no fluorescence) and fused granules (green fluorescence). Simultaneously with the fluorescence changes of spH we monitored loading of granules by FM 4-64 dye. We observed two types of spontaneous exocytotic events. In the events of type-I granules fused with the plasma membrane, the fluorescence of spH rapidly increased. Following the fusion, granules either resealed as indicated by an exponential decrease in spH fluorescence intensity with a time-constant τ of 1.2 ± 0.2 s (mean ± SE, n=9) or persisted opened for the time of monitoring. These granules did not load the FM 4-64 dye. In the events of type-II, after fusion granules loaded with the FM 4-64 dye (t_20-80% 3.0 ± 0.8 s, n=15). Most of fused granules resealed as indicated by a double exponential decay in spH fluorescence intensity with time-constants τ₁ of 1.9 ± 0.5 s and τ₂ of 31.1 ± 5.1 s (n=12), however some granules persisted in an opened state, showing only a slight decrease in spH fluorescence over time. The time-course of spontaneous granule fusion was fast in both types of events (t_20-80% of spH fluorescence <0.5 s) indicating a rapid efflux of protons. We noticed that 4 out of 50 granules fused and resealed more than once, suggesting that these periodical events were due to transient fusion pore openings (flickers). Based on our study we conclude that fusion pore dimension is smaller in events of type-I compared to the events of type-II since it permits the exit of protons but not the entry of large molecules as FM 4-64. We also conclude that the two types of basal exocytotic events undergo different kinetics of resealing.

Email: nina.vardjan@mf.uni-lj.si

Address: Nina Vardjan, Medicinska fakulteta, Inštitut za patološko fiziologijo, Zaloška 4, 1000 Ljubljana