Comparing different fluorescent probes to quantify lipid order alterations during Ca2+ mediated synaptotagmin C2 domain binding to membranes.

Abstract

Changes in membrane order caused by lipids of different acyl-chain saturation or Ca2+ mediated binding of Synaptotagmin 1's C2 domains to the membrane change the equilibrium conformation of the t-SNARE Syntaxin 1a's linker region between SNARE motif and trans membrane domain. A more rigid linker in Syntaxin caused by the disordering of the surrounding lipid bilayer led to more fusion between secretory vesicle membranes and target membranes. This observation suggests a mechanism for Ca2+ stimulated exocytosis in which the Ca2+ sensor Synaptotagmin acts on SNARE proteins through the lipid bilayer. The exact nature of the physical changes in the lipid bilayer that cause the conformational changes in the SNARE protein are not known. Several fluorescent probes have been introduced and applied that are sensitive to one or more environmental properties of the lipid bilayer. We compare the effects of Ca2+ mediated C2 domain binding to model membranes and different acyl-chains on membrane fluidity, order, polarity, and lateral pressure by measuring fluorescence anisotropy, fluorescence lifetime, and general polarity using four different commonly used fluorescent probes, namely NBD, DPH, Flipper TR, and Laurdan. The direct comparisons of these probes in the same systems explore their sensitivities to parameters that are most relevant to changes in SNARE conformation leading to SNARE mediated fusion that have been reported previously.