WE ARE MOVING TO THE UNIVERSITY OF MIAMI, MILLER SCHOOL OF MEDICINE

DEPARTMENT OF PHYSIOLOGY & BIOPHYSICS

We are looking for post-docs or students interested in one of the following projects:

Investigate the dynamics of vesicle docking, priming and fusion dependent on SNAREs and accessory proteins in live cells.

We expect strong interest to investigate membrane fusion using electrophysiological, electrochemical and advance fluorescence imaging techniques.

Skills to bring or to acquire in our lab:

• Cell preparation and cell culture
• 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.

 

Development 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.

We expect strong interest to investigate membrane fusion using biophysical methods and biophysical chemistry.

Skills to bring or to learn in our lab:

• 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.

 

Molecular Dynamics modeling of the SNARE complex and accessory proteins to elucidate the changes in protein interactions and conformations leading to vesicle fusion.

For this position some experience with molecular dynamics simulations is expected. Opportunities to also engage in wet lab work will be available.

Skills to bring or to learn in our lab:

• 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.