Unconstrained lateral diffusion of concanavalin A receptors on bulbous lymphocytes.

Abstract

The lateral diffusion coefficient, D, of concanavalin A receptors and receptor complexes on the surface of lymphocytes and RDM4 Iymphomas is enhanced by several orders of magnitude to D > 5 x IO-9cm2/sec by induction of swelling of the cells to bulbous form. Treatments with concanavalin A or 7-nitrobenz-2-oxa-1,3-diazole-phallacidin induce blebs and the bulbous form. The resulting separation ofthe plasma membrane from most of the F-actin cytoskeleton is accompanied by release of constraints on lateral diffusion of the cell surface receptors, which allows the diffusivity of these glycoproteins to increase nearly to the limit allowed by membrane viscosity.Fluorescence photobleaching methods (1-4) have been providing valuable quantitative information on the diffusibility ofvarious cell surface components within the plasma membranes of living cells in the culture. Recent experiments have established two ubiquitous features (4-9): first, a large fraction ofcell surface proteins are not at all diffusible; second, the diffusivities of the mobile fractions are several orders of magnitude smaller than expected for unrestricted diffusion in a viscous lipid bilayer matrix (10, 11). A "fluid mosaic" membrane model (12) alone does not account for the nondiffusible protein fraction nor for the measured quantitatively slow diffusion.As a result of these observations, several mechanisms intrinsic to cellular organization have been proposed (13-17) to account for the restricted lateral diffusion ofcell surface proteins. A prevailing view suggests that the lateral diffusion of surface proteins is restricted by their interactions with the cytoskeletal elements, primarily microfilaments. Several recent experiments demonstrated that the diffusion coefficients of band 3 proteins on erythrocyte ghosts are greatly enhanced by chemical treatments that disrupt the spectrin-actin cortex (18)(19)(20). However, in cultured cells, the applications of cytotoxic drugs that disrupt cytoskeletal structure have generated only small effects on protein diffusion (6,9,21,22).In this communication, we report experiments on the diffusion of concanavalin A (Con A) receptors on murine spleen lymphocytes (MSL) and lymphoma RDM4 cells that were subjected to a treatment that swells the cells to spherical form and detaches them from the bulk of the F-actin cytoskeleton. We observed that, after treatment with Con A or with a phallotoxin, a small fraction of MSL and RDM4 cells swell to form spherical bulbous cells about twice their original diameter. Fluorescence staining of F-actin with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) (23) demonstrated that the bulk of the F-actin cortical cytoskeleton in these bulbous cells had separated from the cell periphery and remained as mesh tightly wrapped around the nucleus. We measured the lateral diffusion of Con A receptors in the plasma membranes of these bulbous cells by fluorescence photobleaching recovery (FPR) methods (2) and found that most Con A receptors were diffusible with an enhanc...

A localized surface protein of guinea pig sperm exhibits free diffusion in its domain.

“…Compared to N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine, it diffuses at a rate near the predicted theoretical limit for a freely diffusing membrane protein (32)(33)(34) . Similar diffusion coefficients have been measured for rhodopsin in amphibian rod outer segment disks (35,36), and for plasma membrane proteins in membranes in which an underlying cytoskeleton may be modified or is separated from the membrane (37)(38)(39)(40)(41)(42) . It is not known whether a membrane-associated cytoskeleton is present in the posterior tail of guinea pig sperm .…”

Consequences of membrane topography

“…This finding should not be taken as proof that plasma membrane compartmentalization and interaction between plasma membrane components do not occur, but only that topography needs to be rejected as a plausible explanation before claiming that the diffusion is anomalous. There are several examples where cell surface smoothness has been linked to higher diffusion rates [8,11,56], supporting that convoluted and folded membranes cause an underestimation of diffusion rates when interpreted in 2D rather than 3D. Cell topography calls for a fundamental re-evaluation of the large body of work on SPTs as well as on fluorescence correlation spectroscopy (FCS).…”

“…However, these numbers are of the same order of magnitude as the excess plasma membrane. For instance lymphocytes have a highly folded membrane and can be induced to double their diameter suggesting that their plasma membranes accommodate at least four times more membrane than the cell shape suggests [8]. Since diffusion coefficients are generally measured by the mean square deviation, the effect of a twofold underestimation of membrane area per pixel will result in a fourfold underestimation of the diffusion coefficient [9].…”

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

“…These GPMVs show simple, low-curvature geometries of giant unilamellar vesicles and appear free of cytoskeletal constraints. By using fluorophores providing recently characterized membrane fluid phase partitioning behavior (T.B., G. Hunt, E. R. Farkas, W.W.W., and G. W. Feigenson, unpublished data) for lipid phase characterization here, we find that these GPMVs can segregate into multi-micrometer-scale coexisting fluid phases, thereby enabling extension of early cell blebbing studies in our laboratories in the 1980s (15,(18)(19)(20). These results are noted and used throughout this paper.…”