Single Particle Tracking of Plasma Membrane Proteins.

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.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107057/1/edwald_1.pd

Oxygen Depletion Speeds and Simplifies Diffusion in HeLa Cells

AbstractMany cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥15 min, diffusion rates increase by >2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity