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Tracking the SNARE Complex

The protein SNAP-25 is floppy on its own (upper), but folds into a tight paper clip-like structure (lower) when it gets ready to mediate neurosecretion. In an engineered version of the molecule, the paper-clip structure causes energy transfer between two fluorescent proteins, CFP and Venus.

The fusion of secretory vesicles with the plasma membrane is essential for the release of neurotransmitters from neurons and hormones from endocrine cells. These fusion events are catalyzed by the interaction of two proteins on the plasma membrane of the cell, syntaxin and SNAP-25, with a third protein, termed VAMP/synaptobrevin, located on the secretory vesicle. The three proteins, collectively called SNAREs, assemble into a core complex that pulls the vesicle and plasma membrane together by forming a twisted bundle of parallel &aalpha;-helices. The steps involved in the formation of the core complex are enigmatic and represent a potentially important aspect of regulation. Conceivably, this process could contribute to short-term synaptic plasticity.

To understand the mechanism underlying SNARE complex formation, the Almers lab developed a fluorescent SNAP-25 molecule that monitors its association with other core complex components. The modified SNAP-25 molecule, termed SCORE (SNARE Complex Reporter), changes its structure when it associates with the complex, allowing intra-molecular fluorescence energy transfer (FRET) between two portions of the engineered protein. The SCORE protein was as effective as unmodified SNAP-25 in assembling into functional complexes, assembling reversibly when calcium enters the cell during depolarization. Development of this system for tracking SNARE complex formation in living cells will allow investigators to monitor and, potentially, uncover novel mechanisms of regulation. By combining SCORE measurements with evanescent field microscopy, a method developed in the Almers lab to visualize the release of single vesicles, it is now possible to detect the first and final steps in neurosecretion.

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