Lateral diffusion of bacteriorhodopsin and a lipid analogue has been measured in dimyristoylphosphatidylcholine bilayers as a function of temperature, phospholipid/protein (mol/mol; L/P) ratio, and aqueous phase viscosity. The protein lateral diffusion coefficients measured above the temperature at which the lipid gel-liquid/crystalline phase transition occurs (Tc) are combined with previously determined rotational diffusion coefficients to provide a test of the Saffman-Delbrfick equations [Saffman, P. G. & Delbrfick, M. (1975) Procm NatL Acade Sci USA 72,[3111][3112][3113]. Insertion of the diffusion coefficients into these equations enables the protein diameter to be calculated. The value of 4.3 ± 0.5 nm so obtained is in reasonable agreement with the known structure of bacteriorhodopsin. A 12-fold increase in the viscosity of the aqueous phase reduces protein lateral diffusion coefficients by 50%, which is also consistent with the Saffman-Delbruick equations. Both protein and lipid lateral diffusion coefficients decrease with decreasing L/P ratio above the T,. It is argued that, at a high L/P ratio, this effect is probably due to changes in membrane viscosity while, at a low L/P ratio, "crowding" effects (steric restrictions) and protein aggregation become important. When comparing diffusion measurements made in different systems, it is important to take the effect of the L/P ratio into account. When this is done, other published measurements offreely diffusing membrane proteins are in good agreement with the present results and the predictions of the Saffman-Delbruick equations. Below the Ta, the presence of protein enhances diffusion rates. The overall effect is to smooth out the large change in diffusion coefficient that occurs at the T,.It is probable that diffusion plays a crucial role in a number of membrane functions [e.g., receptor-mediated processes (1), electron transfer (2), and photoreception (3)]. Much has been learned about this field by the use of optical techniques developed for measuring diffusion of membrane components (for review, see refs. 4-6). Fluorescence microphotolysis (7) is a versatile means for measuring translational and rotational diffusion in single cells, isolated membranes, artificial membranes, and solution (8-13). Transient dichroism of intrinsic chromophores or triplet probes has provided data on the rotational diffusion of a variety of membrane proteins (14). In the case of triplet probes, rotational diffusion has also been measured by using phosphorescence (15,16), delayed fluorescence (17), and fluorescence-depletion signals (18).A fruitful approach for analyzing parameters that potentially might restrict and regulate mobility in cellular membranes is the study of artificial bilayer membranes. Membranes made from a single lipid species (19, 20) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

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Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It is deduced that both fast and slowly rotating forms of band 3 coexist in the membrane. The equilibrium between these forms is temperature dependent, the slowly rotating species becoming increasingly dominant as the temperature is reduced. Plots of the fractional distribution of the different species against temperature show a marked change of slope at around 37--40 degrees C. The effects are essentially reversible over the range 1--45 degrees C and independent of the presence of the spectrin--actin network. The results could be due to temperature-dependent protein--protein associations mediated either by a protein conformational change or by lipid phase segregation. In further experiments, the cholesterol content of the erythrocyte membrane is varied by incubation with lipid vesicles. No significant changes in the rotational diffusion of band 3 are observed following variation of membrane cholesterol/phospholipid mole ratios over the range 0.34--1.66. This is a surprising result in view of the well-known effects of cholesterol on lipid fluidity.

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Direct physical evidence for the linkage of a band 3 population to the cytoskeleton in the erythrocyte ghost membrane is presented. The rotational diffusion of band 3 proteins was measured by observing flash-induced transient dichroism of a covalently bound eosin probe. After proteolytic release of a 40,000-dalton cytoplasmic segment of band 3 by trypsin, a considerable enhancement in the decay of the absorption anisotropy was observed. Analysis of the data indicates that proteolytic cleavage of band 3 produces a mobile band 3 population which has restricted mobility in the unperturbed membrane due to protein-protein interactions involving the cytoplasmic band 3 moiety. Band 2.1 (ankyrin) or 4.1 or both are likefy to be involved in this interaction because a similar effect on band 3 mobility is observed after low-salt/high-salt extraction of these components. Quantitatively, it is estimated that up to 40% of band3 may be linked to the cytoskeleton. Because the ankyrin-band 3 dimer stoichiometry in the membrane is ap proximately 1:5, only about 20% of band 3 dimers can be directly linked to an rin. The remainder could be explained by the existence of higher oligomers of band 3 linked to single ankyrin polypeptides or by linkages involving other components such as band4.1 or 4.2. Elucidation of the interaction between integral and peripheral membrane proteins is important for the understanding of membrane structure and function. The human erythrocyte membrane has often been used as a model system to investigate both the assembly of cytoplasmic structural proteins at the inner membrane surface (the cytoskeleton) and the possible associations of membrane-spanning proteins with this sub-membraneous network (for a recent review, see ref. 1).On the basis of redistribution of membrane components seen in electron micrographs, a direct linkage between integral and peripheral proteins in human erythrocyte membranes was originally suggested (2-4). On the other hand, the rotational diffusion of band 3, the major membrane-spanning protein of the erythrocyte membrane, was found to be essentially independent of the presence or absence of most of spectrin-actin, the quantitatively dominant components of the cytoskeleton (5, 6). Although not ruling out a direct linkage between band 3 and spectrin-actin, this latter finding pointed toward an alternative model in which the bulk of the integral proteins is considered to be trapped within the interstices of a spectrinactin network (5, 7).With the identification of band 2.1 (ankyrin) as the membrane binding site for spectrin (8-10), important progress has been achieved in understanding the interaction between the cytoskeleton and the membrane. Moreover, it was recently shown that ankyrin binds to band 3 in detergent (11), thus immediately raising the question of whether such an interaction also takes place in the intact membrane.Rotational diffusion of band 3 proteins has been measured by using triplet probes (5, 12, 13). In combination with biochemical manipulations of the m...