Research

Research Projects

We Investigate the dynamics of vesicle docking, priming and fusion dependent on SNAREs and accessory proteins in live cells using electrophysiological, electrochemical and advanced fluorescence imaging techniques.

Current methods include

• Cell preparation and cell culture
• Molecular biology
• Whole cell patch clamp
• TIRF microscopy
• Computer Image analysis
• Electrochemical sensing

One approach combines amperometry using microfabricated electrochemical detector (ECD) arrays with total internal reflection fluorescence (TIRF) imaging to study the molecular mechanism of vesicle fusion and transmitter release in chromaffin cells. A second approach uses whole cell patch clamp capacitance measurements to investigate the functional performance of fluorescently labeled proteins in knock-out mouse embryonal chromaffin cells and the function of otherwise modified proteins involved in vesicle priming and fusion. Amperometric recordings can be performed with a time resolution of a millisecond or less and by averaging fluorescence changes from multiple fusion events, the time of such fluorescence changes relative to the fusion event can be determined with very high precision, not limited by the exposure time used in the fluorescence image acquisition. This has become possible with the time super-resolution approach named Event Correlation Microscopy (ECOM), developed in our lab.

In a second project we develop of a highly innovative technology that will enable experiments to achieve a precise mechanistic understanding of structural molecular rearrangements associated with the fusion of neurosecretory vesicles at the plasma membrane.

Current methods include

• Cell preparation and cell culture
• Subcellular fractionation
• Microfabrication in clean room facility
• TIRF microscopy
• Computer Image analysis
• Electrochemical sensing

The approach combines microfabricated electrochemical detector (ECD) arrays, with reconstituted supported membranes to study fusion of isolated chromaffin granules simultaneously by amperometry and total internal reflection fluorescence (TIRF) imaging. We have previously performed combined ECD and TIRF experiments using intact chromaffin cells and discovered a rapid conformational change in SNAP25 associated with fusion events using a FRET construct incorporating CFP/Venus. Proceeding to the reconstituted system will make it possible to incorporate small labels at arbitrary sites in the SNARE proteins or other accessory proteins, a technology that will make it possible to identify precisely which amino acids in the SNARE complex and accessory proteins move and change distance at specific times during the fusion process. If successful, this technology will enable the experimental identification of the detailed molecular steps in vesicle fusion.

In a third approach we perform Molecular Dynamics modeling of the SNARE complex and accessory proteins to elucidate the changes in protein interactions and conformations leading to vesicle fusion.

Current methods include

• MD simulations using GROMACS
• Conversion of protein complex pdb structures to coarse grained models
• Coarse grained simulations using MARTINI force field
• Atomistic simulations using GROMOS96
• Simulations of membrane self assembly

We have developed approaches to simulate self-assembly of the SNARE complex components in asymmetric membranes with physiological lipid composition and applied these approaches to provide a molecular movie of the formation of a fusion pore by an arrangement of SNARE complexes. The person joining this project will proceed to a more complex step including the accessory proteins complexin, synaptotagmin, Munc-18 and Munc-13, which regulate the function of the SNARE complexes. The ultimate goal is to generate a molecular movie from the activating step of Ca2+ binding to fusion pore formation.