Constrained diffusion or immobile fraction on cell surfaces: a new interpretation.

Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.

Redistribution of plasma membrane proteins by electroosmosis elicits cytosolic calcium response in tumor mast cells.

Activation of mast cells and basophils by binding of ligands that crosslink and micro-aggregate cell surface receptors leads to a series of responses including a phosphoinositide cascade, elevation of intracellular free calcium ([Ca2+]i), morphological changes in the cell plasma membrane, and ultimately, exocytosis of granules containing histamine and other mediators of the allergic response. In rat basophilic leukemia (RBL) cells, a tumor mast cell line, stimulation by immunoglobulin E receptor crosslinking induces these responses. In order to determine whether redistribution or aggregation of cell surface proteins is sufficient to induce a response in these cells without extrinsic crosslinking, we have redistributed cell surface proteins by electroosmotic segregation and looked for second messenger [Ca2+]i responses. Video imaging of calcium ion activity using the fluorescent calcium sensitive dye fura-2 revealed the effects of receptor motion and aggregation induced by application of small (10 V/cm) electric fields. A synchronous, monotonic rise in [Ca2+]i generally occurs within a few minutes after a steady field has been applied, while the redistribution of surface proteins is still in progress. The oscillations in [Ca2+]i characteristic of antigen-stimulated cells are not seen, nor are any effects observed in weak alternating fields (0.02, 60 Hz). The observed rise in [Ca2+]i induced by static electric fields is attributed to perturbation of [Ca2+]i regulation by the large-scale redistribution of membrane constituents induced by surface electroosmosis.

Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents.

The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)-receptor complexes on the surface of rat basophilic leukemia cells increase 2-5x following permeabilization with streptolysin O or digitonin, with little change in their mobile fractions. Swelling by hypo-osmotic treatment in water enhances lateral diffusion of IgE-receptor complexes and raises the mobile fractions to near 100%. In contrast, swelling by treatment with filipin arrests lateral diffusion, although rotational mobility remains unhindered. Lateral mobility of a fluorescent lipid analogue remains unchanged under these conditions. Crosslinking by anti-IgE antibodies redistributes the IgE-receptor complexes into large patches on untreated cells and on cells swollen by permeabilization with streptolysin O or digitonin, but not on cells swollen by treatment with filipin. The results indicate a diversity of effects of the various permeabilizing agents on the mobility of membrane proteins. In particular, treatment with filipin appears to reorganize the plasma membrane into a network of fluid domains on a scale smaller than the bleaching spot size used (approximately 1.5 microns).

Effects of protein concentration on IgE receptor mobility in rat basophilic leukemia cell plasma membranes.

The ability of variations of membrane protein concentrations to modulate the lateral diffusion rate of an exemplary membrane protein has been studied in healthy and osmotically shocked cultured cells of the rat basophilic leukemia cell line, 2H3 subclone. Cell surface protein was redistributed by the method of in situ electrophoresis; exposure to electric fields of 1.25-5 V/cm results in cathodal migration of the majority of the surface proteins on this cell type (Ryan, T. A., J. Myers, D. Holowka, B. Baird, and W. W. Webb. Science [Wash. DC]. 239:61-64). Even in these small fields, the steady-state distribution becomes "crowded" with more than an 80% protein occupancy of accessible membrane area at the cathodal end of these spheroidal cells, and the anodal end becomes significantly depleted. We have employed fringe pattern fluorescence photobleaching with CCD imaging detection to measure lateral diffusion coefficients of the liganded IgE receptor on both crowded and uncrowded regions of individual rat basophilic leukemia cells. We find no significant difference in lateral diffusion rates in these regions. Cells swollen by hypoosmotic stress exhibit faster diffusion overall, with the uncrowded regions having a significantly greater increase in diffusion coefficient than the crowded regions. These results are consistent with the partial or total release of cytoskeletal constraints to membrane protein diffusion induced by osmotic stress.