Summary of the Project

Endocytosis of synaptic vesicles is a key event for maintaining neuronal communication, but the mechanisms regulating this process have been controversial for several decades. We will investigate how synaptic vesicle fusion and retrieval are coupled at the synapse by using techniques that allow direct measurement of these processes combined with visualization of molecular events.

The classical picture of endocytosis at the synapse suggested that this process was analogous to receptor-mediated endocytosis, which is a relatively slow process requiring the coating of the cytoplasmic vesicle surface with a meshwork of clathrin molecules. This picture had to be fundamentally revised when direct measurements of endocytosis in hippocampal boutons and bipolar cells demonstrated that synaptic vesicles can be recycled very fast (~ 1s). Fast endocytosis is coupled to exocytosis because it is also triggered by calcium influx. This information was revealed by the use of fluorescent markers (FM1-43) and capacitance measurements of changes in membrane surface area, demonstrating the importance of using direct methods. These measurements also revealed a second, slower component of retrieval (10-30 s) that predominated after stronger stimulation. What are the molecular mechanisms underlying fast and slow endocytosis of synaptic vesicles? To approach this fundamental question we will begin by characterizing the role of clathrin-mediated endocytosis (CME) because this process has been very well characterized and existing evidence strongly suggests that clathrin is involved in at least some forms of endocytosis at the synapse. We will address the following questions: 1) Are fast and slow modes of endocytosis at the synapse both dependent on CME ? 2) Do fast and slow endocytosis occur at the active zone or at different sites on the plasma membrane? 3) What molecular events underly assembly of a clathrin cage at the surface membrane and then clathrin disassembly after retrieval ? 4) What is the Ca-sensitive step involved in triggering the fast mode of retrieval ? Our approach is informed by the conviction that reliable answers to these questions requires the use of methods that measure events as directly as possible. We will investigate the kinetics of CME at the level of single vesicles by applying high-resolution optical methods, Total Internal Reflection Fluorescence Microscopy (TIRFM) and Standing Wave Fluorescence Microscopy (SWFM), in combination with cell-attached capacitance measurements (CACM). These methods will be applied to isolated synaptic terminals from mouse (bipolar cells and hippocampal CA3 nerve terminals). To visualize release sites and sites of CME we will engineer transgenic and knock-in mice of GFP-clathrin and pHluorin-synaptobrevin. We will measure the kinetics of clathrin cage assembly, its location relative to release sites and its putative modulation by cytosolic calcium or synaptic activity. We will quantify CME and directly correlate these measurements with the amount of membrane endocytosed as assayed by CACM. A key aim will be to establish whether endocytosis at the synapse can occur by a mechanism that does not involve clathrin and determine the functional relevance of this mechanism for synaptic transmission.