Combining fluorescence and electrochemical imaging of chromaffin granule fusion with supported bilayers.

Abstract

The main barrier to a full understanding of the detailed molecular conformational changes leading to fusion pore formation is the lack of a technique that allows to experimentally measure simultaneously and with (sub-)millisecond time resolution specific conformational changes in the fusion machinery and the formation of the fusion pore. To overcome this barrier we have developed microfabricated electrochemical detector (ECD) arrays patterned on microscope coverslips on which a chromaffin cell can be placed such that the cell surface in contact with the coverslip can simultaneously be imaged in total internal reflection fluorescence (TIRF) excitation mode while the released catecholamine molecules are oxidized by the ECDs producing amperometric oxidation current spikes. The amperometric spikes indicate not only the time of fusion with <1ms precision but also the location of the release event. Using SNAP25-based FRET probes we demonstrated a conformational change in the SNARE complex preceding fusion pore formation. However, the fluorescent protein (FP) based tags, are rather bulky and it is thus not clear what the conformational change exactly is. For a clear interpretation, the use of localized small labels will be needed. Labeling of specific residues requires the use of recombinant proteins in a reconstituted system. We therefore develop a technology where labeled recombinant SNARE proteins will be incorporated into a supported membrane that covers ECD arrays such that TIR-FRET imaging of conformational changes can be performed simultaneously with electrochemical detection of individual fusion events of dense core vesicles with the supported membrane. FRAP measurements show that a fluid membrane is indeed formed on the ECDs and docking and fusion of membrane labeled bovine chromaffin granules could be observed. Supported by NINDS (NIH) R21 NS118319.