? We propose experiments that have significance at two levels. First we will continue to develop and prove the effectiveness of our recently developed, transgenically encoded technique for protein photoinactivation in vivo. If we continue to be successful in this arena, it will substantiate this technique and encourage the use of this approach in other transgenic systems. This technique has the potential to be broadly applicable to diverse areas of basic research in cell biology, neuroscience, cancer and stem cell biology. Second, we propose to use our technique of protein photoinactivation to further dissect the molecular mechanisms that control synaptic vesicle endocytosis in vivo. Synaptic vesicle endocytosis is essential for the maintenance of appropriate neural function in the nervous system. It has been hypothesized that rapid, clathrin-independent endocytosis occurs at the neuronal synapse and that this form of endocytosis is necessary to maintain a releasable pool of synaptic vesicles. However, because clathrin mutations are early embryonic lethal it has not been possib]e to eliminate clathrin assembly at a mature neuronal synapse and directly test whether clathrin-independent endocytosis exists. Dominant interfering approaches have been attempted but in no case has it been possible to eliminate clathrin function at a mature synapse. We propose to photoinactivate clathrin heavy chain (Chc) and block clathrin assembly over a timecourse of seconds to minutes. If we observe clathrin-independent endocytosis following photoinactivation we will then be able to study this form of synaptic vesicle endocytosis in isolation, and we will do so in vivo. Regardless of the experimental outcome, we will also be able to use our new tools to pursue a new generation of experiments examining the molecular mechanisms of clathrin-dependent endocytosis in vivo. ? ?
| Heerssen, Heather; Fetter, Richard D; Davis, Graeme W (2008) Clathrin dependence of synaptic-vesicle formation at the Drosophila neuromuscular junction. Curr Biol 18:401-9 |
| Thompson, Scott M; Kao, Joseph P Y; Kramer, Richard H et al. (2005) Flashy science: controlling neural function with light. J Neurosci 25:10358-65 |
