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THE MOLECULAR MECHANISMS OF SYNAPTIC VESICLE RECYCLING
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Quantitative Live Imaging of Vesicle Recycling in vivo. Sequential images of vesicle recycling imaged live at the Drosophila NMJ. Nerve stimulation induces an increase in pH-GFP fluorescence associated with vesicle release that is followed by a slow decline in fluorescence associated with vesicle endocytosis.
for review see:
Marek and Davis, 2002
Poskanzer et al., 2003 |
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| Project Summary |
| Many of the molecular players that control synaptic vesicle endocytosis and recycling have been identified biochemically. A remaining challenge is to understand how these molecules are organized into a highly efficient molecular machine capable of high fidelity compensatory vesicle endocytosis. Standard genetic approaches have been valuable, but are also limited in many ways. For example, proteins that are required for vesicle endocytosis such as clathrin are also essential for cell viability, preventing a standard genetic analysis. Other proteins participate in both exocytosis and endocytosis, and standard genetic approaches are unable to dissociate the exocytic from endocytic functions for these molecules. Therefore, we have developed new technologies for the acute disruption of protein function, in vivo, using light (Marek and Davis, 2002; Poskanzer et al., 2003). We are combining these tools with quantitative live imaging of vesicle recycling. These new tools are allowing us to dissect the function of molecules in the synaptic vesicle cycle with spatial and temporal resolution of light. |
I. Quantitative live imaging of synaptic vesicle endocytosis. pH-sensitive variants of GFP can be used to image the exocytosis and endocytosis of synaptic vesicles (Figure 1). |
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| Figure 1. Quantitative live imaging of vesicle recycling. pH sensitive GFP is attached to the luminal domain of a synaptic vesicle protein and expressed in vivo. In the acidic lumen of a synaptic vesicle, the GFP is quenched. Upon fusion of the vesicle with the plasma membrane (exocytosis) the GFP is exposed to the higher pH of the synaptic cleft and fluorescence increases. Upon vesicle reformation, the GFP remains fluorescent and is subsequently quenched during the reacidification associated with the loading of neurotransmitter into the synaptic vesicle. The rate limiting step is the endocytosis of the vesicle. Therefore, the change in GFP fluorescence is a quantitative measure of vesicle fusion and subsequent endocytosis. |
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| Figure 2. Quantitative imagine of synaptic vesicle release and recycling. At left are a series of time-lapse images showing a typical stimulus-dependent increase in fluorescence (exocytosis) followed by a slower decrease in fluorescence (endocytosis). At right, fluorescence changes from seven synapses are quantified and averaged. This type of data allows us to determine the kinetics of synaptic vesicle endocytosis in vivo. From Poskanzer et al., 2003. |
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| II.FlAsH-FALI; A Transgenically Encoded Method for Specific Protein Photoinactivation. We have developed a transgenically encoded, highly efficient method for protein photoinactivation based on fluorophore-assisted light inactivation (FALI) (Marek and Davis, 2002; Poskanzer et al., 2003). We have demonstrated that this technique is specific and highly efficient, allowing protein photoinactivation to be achieved in seconds with the spatial precision of light. To do this we have tagged the synaptotagmin I protein (Syt I) with a tetracysteine motif (SytI 4C) that covalently binds the membrane-permeable fluorescein derivative 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH), developed in Roger Tsien's laboratory (Griffin et al.,1998; Gaietta et al., 1999). Photoinactivation of Syt I is able to severely impair synaptic vesicle release in seconds (See Marek and Davis, 2002). We have performed several experiments demonstrating that FlAsH photoinactivation is specific to the SytI molecule at the synapse (see Marek and Davis, 2002). In one such control we demonstrate that FlAsH photoinactivation of Syt I does not alter sucrose evoked release. Sucrose evoked release is SNARE dependent but SytI independent (Aravamudan et al., 1999; Geppert et al., 1994). Thus, SNARE-mediated vesicle fusion remains intact, even after photoinactivation of SytI4C. |
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| Figure 3. Rapid and Specific FlAsH Photoinactivation of SytI4C. (A) FlAsH-photoinactivation of Syt I4C rapidly impairs evoked transmitter release. Photoinactivated synapses in 0.5 mM calcium (open circles) show complete elimination of evoked responses within 30 s. Increasing extracellular calcium to 1.0 mM calcium in the photoinactivated animals (open squares) reveals a persistent evoked release similar to that observed in sytI-null animals. Identically treated wild-type animals (closed squares) show no significant effect during constant illumination. (B) A representative trace from a FlAsH photoinactivation recording. Vertical lines represent individual EPSPs at this condensed timescale. (C) Hypertonic release is normal after FlAsH-FALI of Syt I. The sucrose period is indicated by the horizontal bar. From Marek and Davis, 2002. |
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| Using this new FlAsH-FALI technology, we have been able to dissect the exocytic versus endocytic functions of synaptotagmin I (Poskanzer et al., 2003). By photoinactivating Syt I after vesicle fusion but prior to vesicle endocytosis we show that Syt I is essential for vesicle re-formation (Poskanzer et al., 2003). This conclusion has recently been supported by work at vertebrate central synapses (Nicholson-Tomishima and Ryan, 2004). We are now extending this work to dissect the functions of other essential synaptic proteins during synaptic vesicle recycling. |
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