Size And Acylation Influence The Lateral Mobility Of Plasma Membrane Proteins In Live Cells

Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. Using single fluorescent particle tracking, we quantify diffusion parameters of a large panel of fluorescent fusion membrane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated or non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, truncated haemagglutinin, and β2 adrenergic receptor) as well as three proteins anchored with lipid moieties (GPI, palmitoyl and myristoyl, or geranylgeranyl). We present an analysis that utilizes Brownian simulations to aid in interpreting heterogeneity. With the exception of two of the palmitoylated transmembrane proteins, diffusion of all constructs is unconfined and consistent with Brownian motion at 37°C at the time-scales investigated (20 ms-1 sec). We explore the contributions of lipid mixing to confinement by modulating cholesterol levels and through the use of biochemical perturbations that affect the temperature of the immiscibility phase transition in isolated plasma membrane vesicles. Among our findings is that diffusion and confinement are highly temperature sensitive. Furthermore, our results indicate a complicated size-dependence of diffusion suggesting that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a biological context or due to labeling techniques. Overall, we explore the contribution of phase-mediated membrane heterogeneity to protein mobility while highlighting several factors that can complicate the interpretation of lateral diffusion data.