Spatially resolving light detectors allow, with proper calibration, quantitative analysis of the variations in two-dimensional intensity distributions over time. An ultrasensitive microfluorometer was assembled by using as a detector a microchannel plate-intensified video camera. The camera was interfaced with a software-based digital video analysis system to digitize, average, and process images and to directly control the timing of the experiments to minimize exposure of the specimen to light. The detector system has been characterized to allow its use as a photometer. A major application has been to perform fluorescence recovery after photobleaching measurements by using the camera in place of a photomultiplier tube (video-FRAP) with the goal of detecting possible anisotropic diffusion or convective flow. Analysis of the data on macromolecular diffusion in homogenous aqueous glycol solutions yielded diffusion constants in agreement with previous measurements. Results on lipid probe diffusion in dimyristoylphosphatidylcholine multibilayers indicated that at temperatures above the gel-to-liquid crystalline phase transition diffusion is isotropic, and analysis of video-FRAP data yielded diffusion coefficients consistent with those measured previously by using spot photobleaching. However, lipid probes in these multibilayers held just below the main phase transition temperature exhibited markedly anisotropic diffusive fluxes when the bleaching beam was positioned proximate to domain boundaries in the P A' phase. Lipid probes and lectin receptor complexes diffused isotropically in fibroblast surface membranes with little evidence for diffusion channeled parallel to stress fibers. A second application was to trace the time evolution of cell surface reactions such as patching. The

The lateral mobility of the lipid analog N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine and of the integral protein glycophorin in giant dimyristoylphosphatidylcholine vesicles was studied by the photobleaching technique. Above the temperature of the chain-melting transition (Tm = 23 degrees C), the diffusion coefficient, Dp, of the protein [Dp = (4 +/- 2) X 10(-8) cm2/s at 30 degrees C] was within the experimental errors equal to the corresponding values DL of the lipid analog. In the P beta 1 phase the diffusion of lipid and glycophorin was studied as a function of the probe and the protein concentration. (a) At low lipid-probe content (cL less than 5 mmol/mol of total lipid), approximately 20% of the probe diffuses fast (D approximately equal to 10(-8) - 10(-9) cm2/s), while the mobility of the rest is strongly reduced (D less than 10(-10) cm2/s). At a higher concentration (cp approximately 20 mmol), all probe is immobilized (D less than 10(-10) cm2/s). (b) Incorporation of glycophorin up to cp = 0.4 mmol/mol of total lipid leads to a gradual increase of the fraction of mobile lipid probe due to the lateral-phase separation into a pure P beta 1 phase and a fraction of lipid that is fluidized by strong hydrophilic lipid-protein interaction. (c) The diffusion of the glycophorin molecules is characterized by a slow and a fast fraction. The latter increases with increasing protein content, which is again due to the lateral-phase separation caused by the hydrophilic lipid-protein interaction. The results are interpreted in terms of a fast transport along linear defects in the P beta 1 phase, which form quasi-fluid paths for a nearly one dimensional and thus very effective transport. Evidence for this interpretation of the diffusion measurements is provided by freeze-fracture electron microscopy.

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Glycophorin A is one of the major integral proteins in erythrocyte membranes. We will report on the reconstitution of Glycophorin A into artificial phosphatidylcholine membranes (DMPC) in the concentration range between O,i and i0 mol %o proteins. The influence of the protein on the structure and the dynamics of the lipid matrix has been investigated by the following techniques: i) Electron paramagnetic resonance (EPR) and calorimetry (DTA).We observed a slight shift of the main phase transition temperature of about 3o. The order degree of the lipid fatty acid chains shows a minimum at a molecular content of 0,7 %o Glycophorin. 2) Fluorescence recovery after photobleaching (FRAP) and excimer techniqueThe lateral mobility of Glycophorin and lipid analoges is determined. Zt shows a quite complex dependence from the protein/lipid molar ratio. The "pretransition" of the lipid matrix is clearly more important for the lateral diffusion of Glycophorin A than the main transition. For comparison excimer-technique was applied to determine the lipid mobility by using the lipid analogue pyren lecithin. 3) Energy transferGlycophorin and lipids were labelled with two fluorophores. Energy transfer is measured in different experiments between Glycophorins as well as protein to lipid transfer. Lipid to lipid transfer experiments serve as standard of statistical distribution. At 0,8 %0 is a local maximum of protein-protein ET and a minimum of lipid protein ET. 4) ElectronmicroscopyThe experiments yield information about the surface texture of individual lipid bilayers. Glycophorin seems to be preferentially located in defect lines and -1/2 defects of the P~'phase. Higher concentration of protein leads to a suppression of the P~'phase, but by lowering the temperature the PB'phase reappears. Above 1%0 and at low temperatures particles are observed in the defects of the PB' phase. Glycophorin blocks the pretransition to the L~'phase. We can differenciate three regions of concentration O -O,8 , 0,8 -4 , 4 -i0 mol %0. 0,8 ~o is a distinguished point. The interpretation will be given in terms of aggregational and conformational changes of the glycophorin molecules, carbohydrate-lipid interactions, defect of the lipid matrix, long ranging proteinprotein lipid mediated interactions.

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