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Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5μm 1/e2 radius. The effective spot area represents only 1/500 of the total surface of an 8-μm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100molecules. This limits the precision and reproducibility of FPR measurements. We describe a method for interferometric fringe pattern FPR that permits simultaneous interrogation of the entire cell’s surface. Fringe patterns are generated interferometrically within the optical path of an FPR system. Methods for interpreting fluorescence recovery kinetics on cells and for determining the protein mobile fraction are presented. With fringe FPR, the murine major histocompatibility complex class II antigen I-Ak expressed on M12.C3.F6 cells has 100-fold improved fluorescence signals relative to spot FPR, with corresponding improvements in signal-to-noise ratios of recovery traces. Diffusion coefficients (± standard deviation) of (2.1±0.4)×10−10 and (1.8±1.0)×10−10cm2s−1 with corresponding mobile fractions of I-Ak of 66.1±7.8% and 63.4±18.0% were obtained by fringe and spot methods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be substantial variation from cell to cell in protein dynamics, and this method may permit the assessment of such variation.

Tyrosinase (o-diphenol:O2-oxidoreductase; EC 1.10.3.1) is present in the epidermis of Rana pipiens pipiensas an inactive proenzyme that can be activated by a variety of endopeptidases, chymotrypsin being the most effective. This proenzyme has been purified to homogeneity as judged by polyacrylamide gel electrophoresis.

The critical micelle concentration (CMC) for bile salts or other surfactants is defined as that solute concentration at which appreciable changes in such phenomena as light scattering, surface tension, or solubilization of other organic molecules occur, these changes indicating appearance of surfactant aggregates. The CMC thus reflects hydrophobic interactions of the surfactant with itself. The self-association of hydrophobic molecules resembles the partition of a solute into the lipophilic phase in reversed-phase high performance liquid chromatography (RPLC): Both processes can be considered as transfers of a molecule from an aqueous to a lipophilic medium. The critical micelle concentration of a particular bile salt, being a measure of its hydrophobic self-association, should therefore be correlated with its Chromatographic mobility since they are fundamentally related phenomena. Experimentally, significant correlations between these quantities are obtained, both for bile salts andn-alky1 sulfonates, and only microgram amounts of sample are required for RPLC measurements. Among three homologous series of bile salt surfactants, CMC values predicted from RPLC measurements agree, within a standard error of 7%, with CMC values determined directly. This suggests the applicability of reversed-phase liquid chromatography to the micro-scale determination of critical micelle concentrations of bile salts,n-alkyl sulfonates, and other homologous series of surfactants.

Luteinizing hormone receptors (LHR) are G protein-coupled membrane proteins with important functions in reproduction. We have recently shown that rat and human LHR, upon binding human chorionic gonadotropin (hCG), translocate into plasma membrane compartments of low buoyant density (rafts). This translocation depends on a functional hormone-receptor complex. After binding hCG, LHR become unresponsive to further stimulation by additional hormone. This phenomenon is termed receptor desensitization and is a common regulatory mechanism among membrane receptors. Previous studies indicate that desensitized LHR interact with membrane proteins like β-arrestin-1 and cluster into large, microscopically-visible structures that diffuse slowly in the membrane for several hours. Not until these large structures disperse are LHR again responsive to hormone, as indicated by a rise in the intracellular second messenger cAMP. We examined whether human LHR redistribute into compartments of low buoyant density during desensitization. About 46% of receptors appeared in rafts immediately following desensitization and by 2 hours that number increased to 70%. By 5 hours, when cAMP levels increase in response to hormone challenge, only 9% of receptors were found in rafts. These findings suggest that the localization of human LHR in rafts may be a requirement for receptor desensitization. To further examine the role of receptor compartmentalization in LHR desensitization, we used single particle tracking methods to monitor the lateral diffusion of individual LHR and the size of plasma membrane compartments accessed by receptors on viable cells. Preliminary results indicate that desensitized human LHR on CHO cells become confined in membrane compartments with an average diameter of 84 ± 39 nm. The desensitized LHR remain confined in small diameter compartments for several hours. These compartments are significantly smaller than the 199 ± 79 nm diameter regions occupied by untreated receptors. It is thought that the cytoskeleton plays a role in restricting hormone-treated receptors within small compartments, and this may be true for desensitized receptors as well. Possible membrane models to explain this behavior include compartmentalization by cytoskeletally-anchored proteins that serve as protein fences. This research was supported by CHE-0628260 (NSF), AG030230 (NIH) and RR023156 (NIH).(poster)