Temperature Dependent FCS Shows Mammalian Cell Membrane is not Poised near Miscibility Transition Temperature

Abstract

Immiscibility of liquid mixtures occurs when Gibb's free energy of the separated state is more favorable to that of homogeneous state of the system. Immiscibility is known to be taking action in various lipid mixture membrane systems too. Studies examining macro scale phase separations in ternary lipid mixtures and mammalian membrane blebs have shown that domain separation can also sort membrane components by differential chemical potentials. This behavior is commonly extended to native cell membranes hypothesizing existence of nano-scale heterogeneous domains but actual evidence of this link has been vague. Hence where in the phase diagram the live cell membrane is poised would be a very important question in terms of thermodynamics even considering the possible non-equilibrium perturbation to the membrane by actin cytoskeleton or active exchange of components etc. Temperature dependent fluorescence correlation spectroscopy of fluorescent labeled lipids shows a consistently linear trend of mobility as a function of temperature for several different types of lipids. Supported by Monte-Carlo simulation on random walking particles under different states of the system, it shows cell membrane doesn’t experience effective miscibility transition well below the physiological temperature. Fluorescence lifetime study with DiI, a known local viscosity reporter, confirms this trend, and shows a clear difference in lifetime between living cell membranes and the blebs derived from them. This also shows even though the membrane blebs are directly induced from the native cell membrane, they may not be under the identical environment which would also suggest two systems being at different positions in the phase diagram.