Real-time TIRF imaging of exocytosis in 3T3-L1 adipocytes stably expressing mCherry fused to human adiponectin

Abstract

AbstractAdiponectin is a peptide hormone abundantly released from adipocytes, and its reduced circulating levels have a central role in obesity-related diseases, such as type 2 diabetes, insulin resistance and cardiovascular disease. Adiponectin is secreted by regulated exocytosis of vesicles belonging to a pre-formed vesicle pool, in response to the appropriate stimulatory signal. Traditional molecular biology and imaging techniques lack spatial and temporal resolution to visualize and quantify the exocytosis dynamics of adiponectin-containing vesicles. Here we generate 3T3-L1 adipocytes stably expressing mCherry fused to human adiponectin and we employ total internal reflection fluorescence (TIRF) microscopy in real-time to visualise and quantify exocytosis of the fusion protein. The TIRF recordings show that an intracellular elevation of either cAMP or Ca2+, mediators known to induce adiponectin exocytosis, lead to rapid disappearance of fluorescent vesicles and we interpreted this as exocytosis events. Secretion measurements confirm that the human adiponectin-mCherry fusion protein is secreted to the surrounding solution upon stimulation with agents that elevate intracellular cAMP or Ca2+. We conclude that the fusion protein appear to be directed to vesicles belonging to the endogenous adiponectin vesicle population and that the targeted vesicles are secreted by signals known to stimulate adiponectin exocytosis. Thus, the mCherry-adiponectin-expressing cells are useful for investigations of the adiponectin vesicle exocytosis process in adipocytes, at a level of detail that has not been possible previously.