tSNAREs in the Spotlight

Intercellular communication relies on signal molecules stored in membrane bound compartments called vesicles and released when the vesicle fuses with the plasma membrane. The protein machinery needed for fusion is well established, however its temporal and physical organization is poorly understood. SNARE proteins – two target-SNAREs on one vesicular-SNARE are brought together and ‘zip-up’ to allow lipid mixing between vesicle and membrane, opening a ‘fusion pore’ for signal release. One key question is how the tSNAREs are organized before fusion– do they form a binary target complex, and what regulates it? Fluorescence Correlation Spectroscopy is an excellent way to answer this question. It allows the measurement of molecular diffusion on a millisecond timescale. Cell membranes containing fluorescently labelled tSNAREs are illuminated at a single point (less than a femtolitre in volume). As fluorophores through the beam the detected light intensity varies depending on the speed the molecules move at. These characteristics can be extracted through a process of correlation. When red and green labelled tSNAREs interact and move together a peak appears at the same time in both traces rather than in one or the other. This provides an assay with which interactions can be monitored in the presence of various other components of the system.