Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

Abstract

While gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. Dynamin, a mechanoenzyme, has been however previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse, and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles, and dynamin inhibition, by siRNA knockdown or treatment with a dynamin GTPase inhibitor, dynasore, increased the number and size of gap junction “buds” suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, however the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicles fissions/hour were reduced in the dynamin inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is critical to elucidating the relationship between gap junctions, membrane regulation and cell behaviors.