Elliot Elson

Yanfei Jiang1, Kenneth M. Pryse1, Artem Melnykov1, Guy M. Genin2, and Elliot L. Elson1

1.Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis. 2. Department of Mechanical Engineering, Washington University in St. Louis

Molecular clusters in cell membranes such as rafts, cell-matrix adhesions, and synapses are important for cellular signaling and establishment of cellular morphology. An interesting and challenging problem is to understand how these clusters arise. Lipid bilayer membranes that contain high and low melting lipids that undergo phase separation to form distinct membrane domains provide useful test systems.  Membranes composed of DLPC (12:0, Tm = -2o C) and DSPC (18:0, Tm= 54o C) provide a good example.  Although FCS is a plausible approach to study these phase systems, uncertainties arise due to weak dependence of the diffusion coefficients of phase domains on their sizes and difficulty due to relatively modest differences in partition coefficients for phase-specific fluorescent lipid analogues. We have instead used inverse Fluorescence Correlation Spectroscopy (iFCS) [1]. In iFCS measurements high concentrations of small molecule indicator fluorophores provide a constant baseline fluorescence. As much sparser nonfluorescent dark particles diffuse into or out of the observation volume, they displace a fraction of the indicator molecules proportional to their size, and so generate measurable negative fluorescence fluctuations [2, 3].  As for FCS, the stochastic fluctuations are analyzed statistically, using a fluorescence fluctuation autocorrelation function, G(t), which provides the diffusion coefficient of the dark domains.  In contrast to FCS, G(0) in iFCS depends both on the size of the dark particles and on their average number in the observation volume. We have augmented iFCS with an analysis of moments of fluorescence fluctuations and used it to measure stages of phase separation. With the assistance of a numerical model for the phase separation [4] we have observed two different pathways for the growth of phase domains. In one, nanoscopic gel domains appeared first and then gradually grew to micrometer size. In the other, the domains reached micrometer size quickly, and their number gradually increased.

1- Jiang, Y., et al., Investigation of Nanoscopic Phase Separations in Lipid Membranes Using Inverse FCS. Biophysical Journal, 2017.112(11): p. 2367-2376. 2- Wennmalm, S., et al., Inverse-fluorescence correlation spectroscopy. Anal Chem, 2009. 81(22):p.9209-15. 3- Wennmalm, S. and J. Widengren, Inverse-fluorescence cross-correlation spectroscopy. Anal Chem, 2010. 82(13): p.5646-51.4- Frolov, V.A., et al. "Entropic traps" in the kinetics of phase separation in multicomponent membranes stabilize nanodomains. Biophys J, 2006.91(1): p.189-205.