Single Particle Tracking of Plasma Membrane Proteins.

[摘要] Palmitoylation is important to the function and trafficking of many proteins. As the only reversible posttranslational lipid modification, it is thought to facilitate signaling by dynamically targeting proteins to the necessary membrane fractions. This has been shown for membrane-associated proteins, but the role of palmitoylation for transmembrane proteins is less clear. It has been proposed that palmitoylation targets transmembrane proteins to membrane subdomains often termed ;;lipid rafts’. In this work, we test the hypothesis that palmitoylation affects the diffusion dynamics of transmembrane proteins and propose that this could be a means to modulate protein function. Using single fluorescent particle tracking, this work quantifies diffusion and confinement parameters of a large panel of fluorescent fusion membrane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated and non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, truncated hemagglutinin, and β2 adrenergic receptor) as well as three proteins anchored with lipid moieties (glycophosphatidylinositol (GPI), palmitoyl and myristoyl, or geranylgeranyl). We present a method of analysis that uses Brownian simulations to aid in identifying heterogeneity. Among our findings is that lateral diffusion in a photoprotective hypoxic imaging buffer is Brownian and vastly simplified in comparison to non-hypoxic imaging buffer, suggesting possible cytoskeletal remodeling under hypoxic conditions. In both hypoxic or normoxic imaging conditions, lateral diffusion is strongly size-dependent for smaller probes, consistent with findings in model membranes. Thus our results indicate that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a biological context or due to labeling techniques. Differences in lateral diffusion were not significant at 37°C when comparing otherwise identical transmembrane proteins with and without palmitoylation sites, though the proteins differentiate themselves at lower temperatures. This suggests that palmitoylation does not modulate transmembrane protein function by altering lateral diffusion under physiological conditions.

[发布机构] University of Michigan

[效力级别] Membrane Biophysics

[关键词] Single Particle Tracking;Membrane Biophysics;Plasma Membrane Structure and Assembly;Biological Chemistry;Science;Chemical Biology