Biophysical characterization of styryl dye-membrane interactions

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.