Investigating Protein Dynamics at Sites of Exocytosis in Live Cells

Abstract

Exocytosis is the cellular process in which cytoplasmic membrane bound vesicles fuse with the plasma membrane and release their contents into the extracellular space. Calcium triggered exocytosis is critical for many physiological functions, including release of neurotransmitters by neurons, and secretion of hormones by endocrine glands. Though the basic steps and the key regulatory proteins involved in this process are known, the temporal and spatial dynamics of these molecules at the sites of exocytosis remain unclear. Here, we use total internal reflection fluorescence microscopy to visualize protein dynamics at individual synaptic-like microvesicles in live PC12 cells (a rat adrenal chromaffin-derived cell line). We employ two-color imaging to simultaneously observe vesicles undergoing fusion (using a vesicular neurotransmitter transporter as the marker), and the dynamics of various exocytic proteins at these sites of fusion, before, during and following fusion. Our experiments reveal that while many exocytic proteins such as the SNARE proteins, SNARE modulators and Rab proteins are already present at the sites of fusion, the SNARE modulator tomosyn and the Rab proteins, Rab3A and Rab27A, leave these sites soon after fusion. Interestingly, we find that the endocytic coat protein clathrin is recruited to sites of fusion several seconds following fusion, suggesting spatial and temporal correlation between exocytosis and compensatory endocytosis. Our findings provide insights into the dynamics of key mediators of exocytosis and endocytosis during the moments surrounding fusion in live cells, and advance our understanding of the regulatory roles of these various proteins.