CaPeBlE

The interaction of a cell with its environment occurs through the plasma membrane. To regulate the shape and composition of their plasma membranes, and to internalize nutrients and signaling factors, eukaryotic cells use endocytosis, the formation of vesicles from the plasma membrane. Clathrin mediated endocytosis (CME) is the most widely used form of endocytosis. The dynamics and molecular mechanisms of CME, and its role in a number of cellular functions, has been the subject of intense study. A vast number of proteins and interactions implicated in this process have been identified, but the dynamics of these interactions is largely unknown. Our interdisciplinary team of biologists and chemists will develop new tools to visualize and interfere with selected key interactions leading to clathrin coated vesicle (CCV) formation. First, we will synthesize photoactivatable, or caged, peptides to block the interaction between the proline rich domain of dynamin, a protein essential for CME, and the SH3 (Src homology 3) domain of its partners, such as amphiphysin. This will block CME in live cells in a spatially and temporally controlled way, which we will test with live cell imaging of CCV formation. Second, we will construct pairs of endocytic proteins fused with fluorescent proteins to visualize their interaction by Förster Resonance Energy Transfer-Fluorescence LIfetime Microsopy (FRET-FLIM) imaging in live cells. We will connect the timing of this interaction to the stages of endocytic vesicle creation. This set of experiments will provide unprecedented details on the dynamics of molecular mechanisms of CME. Moreover, the caged peptides developed in this project will be usable in a variety of cellular contexts. In particular, CME is essential for the expression of synaptic long term depression (LTD), a neuronal substrate of certain forms of learning and memory. We will perform electrophysiology patch clamp experiments to determine with the caged peptide where and when CME is required for LTD, and whether endocytosis contributes to the synapse specificity of LTD. We anticipate that these tools, once made available to the scientific community, will clarify the involvement of CME in a number of cellular functions.