Hormones and neurotransmitters are stored in endocrine and nerve cells in small packets called vesicles. When a cell is stimulated, secretion occurs and the vesicles empty their contents out of the cell. However, secretion will occur only if the vesicles have undergone biochemical changes called priming. Priming, therefore, determines how much and how fast secretion can take place. Increased understanding of priming could lead to new treatments in devastating diseases such as type 2 diabetes, and Parkinson's, in which too little secretion occurs. Furthermore, it may help increase the output of biotechnology products that are secreted from cells in culture. I propose to develop a novel method to allow one to see individual primed vesicles. The method is based on previous work of the Chow laboratory that showed that the protein complexin is critical for priming. Complexin binds tightly to the SNARE complex, a hetero-oligomer of three proteins that together triggers the fusion of vesicle and plasma membranes during secretion. By labeling complexin with a fluorescent tag, we will be able to identify the vesicles (labeled with a separate fluorescent tag) that have complexin bound to the vesicle-associated SNARE complex. We will use total internal reflection fluorescence microscopy (TIRFM) to detect co-localization of tagged complexin with primed vesicles and we will use this assay to assess and differentiate the roles of other proteins (Munc 13 and CAPS) in modulating priming kinetics. In addition, we will also address the mystery as to how many SNARE complexes (using tagged complexin as the reporter) are physically associated with a single vesicle. This will be accomplished by using a new super-resolution method called fluorescence photoactivation localization microscope (FPALM). FPALM increases optical resolution to near-molecular dimensions (30-50 nm has been accomplished) in living or fixed cells. The combination of the new primed vesicle marker and FPALM will speed up identification of other proteins involved in priming and provide a means to study detailed protein-protein binding kinetics. It will give insights into possible approaches to treat diseases in which too little secretion occurs. Defining the roles of proteins involved in vesicle priming using our novel approach may lead to developments of new drugs or other treatments to compensate for altered expression of proteins involved in vesicle priming. In addition, the knowledge gained from research in understanding how to manipulate vesicle priming may be used in several biotechnology applications where production of secreted products is the limiting step.
| Rohan, Joyce G; Citron, Y Rose; Durrell, Alec C et al. (2013) Light-triggered modulation of cellular electrical activity by ruthenium diimine nanoswitches. ACS Chem Neurosci 4:585-93 |
| Lin, Ming-Yi; Rohan, Joyce G; Cai, Haijiang et al. (2013) Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems. J Physiol 591:2463-73 |
