Analysis of Newcomer Insulin Secretory Granule Movement by Newly Developed Technique, Variable TIRF Real-Time Imaging System
Abstract Number: 188-OR
Authors: MICA OHARA-IMAIZUMI, YOKO NAKAMICHI, YOSHIHIKO WAKAZONO, TAKASHI SAKURAI, SUSUMU TERAKAWA, SHINYA NAGAMATSU.
Institutions: Mitaka, Tokyo, Japan; Hamamatsu, Shizuoka, Japan.
Results: The use of internal reflection fluorescence microscopy (TIRFM), which allows fluorescence excitation within a restricted domain close to the plasma membrane, has permitted us to observe single insulin granules undergoing exocytosis. By this system, we could observe and analyze a first phase of insulin exocytosis, where a small portion of insulin granules docked at the plasma membrane were fused with plasma membrane. However, as long as we use this original system, it is difficult to reveal the mechanism underlying the second phase insulin release, where newcomer insulin granules are translocated from inner pool, targeted to, and released from, the plasma membrane. In order to overcome this problem, we have innovated the variable TIRF real-time imaging system. V-TIRF permitted to manipulate the penetration depth of evanescent field by automatically switching the angel of incidence, so that we could simultaneously observe in real-time the different regions of 500 nm and within 100 nm from cell surface. Here, using v-TIRF technique, we imaged and analyzed the real-time motion of single insulin granules from cytosol inner pool to the plasma membrane, finally fusion process. The results showed that there are two pools of insulin secretory granules docked at the plasma membrane and existed in the cytosol located 500 nm from the cell surface. V-TIRF revealed that glucose stimulation evoked the translocation of insulin granules from inner pool to the plasma membrane, and fusion of granules with cell membrane was occurred within 50 ms after reaching the cell surface. Furthermore, the experiments using recombinant adenovirus encoding actin-EGFP treated b cells showed the close association of insulin granule intracellular movement with actin-network. Thus, v-TIRF real-time imaging system is a valuable technique to reveal the mechanism of intracellular vesicle trafficking.