Dynamics of Crowding-Induced Mixing in Phase Separated Lipid Bilayers

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (L_o)-liquid disordered (L_d) phase separated lipid bilayers when the following particles of increasing size bind to either the L_o or L_d phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu²⁺ chelating mechanism, of lipids that specifically partition into either the L_o phase or L_d phase. The degree of steric pressure was controlled by varying the size of the bound particle (10-240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed L_o phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.