Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.

Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (diI(3)-LDL). Fluorescence photobleaching recovery and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10°C, <20% of the LDLreceptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor-internalization-defective J. D. cells GM-2408A. At 21 ° and 28°C, the diffusion coefficients of the LDL-receptor complex were 1.4 and 4.5 x 10 -11 cm2/s with diffusible fractions of ~75 and 60%, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5 X 10 -8 cm2/sec at 22°C. On blebs induced in GM-2408A cell membranes, the diI(3)-LDL receptor complex diffusion coefficient increased to ~10 -9 cm2/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD-phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on lateral diffusion observed on blebs. This work shows that the internalization defect of 1. D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDUreceptor diffusion may not limit LDL internalization in normal human fibroblasts.Low density lipoprotein (LDL), like insulin and epidermal growth factor (EGF), is one of a class of proteins that binds to a specific high-affinity cell surface receptor (2, 3) and is then internalized by the cell at a clathrin-coated area of membrane, a coated pit (4, 5). In normal human fibroblasts, >70% of the LDL receptor (LDL-R) population appears localized in coated pits even before LDL binding occurs (6). Thus, it is unclear whether diffusion to a coated pit on the membrane by either the bare LDL receptor or LDL bound to the LDL receptor, the LDL-receptor complex (LDL-RC), is a necessary step in the regulation of cholesterol synthesis and degradation. In contrast, the insulin and EGF-receptor complexes are reported to diffuse and form dusters before internalization at coated 846 pits (8, 9). The mobility of a ligand-receptor complex can be studied by fluorescence photobleaching recovery (FPR) by monitoring the characteristics of the relaxation of ...

The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL . Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptornegative human fibroblasts at 4°C or 37°C . It is internalized rapidly at 37°C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluoresceinlabeled antibody or directly by dil(3) fluorescence . Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory .

Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP been identified on the surface of many fibroblastic cells (1, 2). The amount of this protein decreases substantially after transformation (2). Designated cell surface protein (CSP) (3) or large, external, transformation-sensitive (LETS) protein (4), this protein has been isolated from chick embryo fibroblasts (CEF), in which it constitutes 3% of the total cell protein (1, 3). CSP agglutinates erythrocytes, suggesting that it may play a role in cell adhesion (5). This possibility is reinforced by the observation that addition of exogenous CSP to transformed cells partially restores the morphology, adhesiveness, and parallel alignment of cells that are typical of normal fibroblasts, although the added CSP has no effect on growth control (6). The effects of CSP on cell morphology have recently been interpreted as resulting from its enhancement of cell adhesion (1, 6).This report describes an investigation of how CSP binds to the cell surface, focusing particularly on its lateral mobility, its interactions with the lipid phase of the membrane and with various other membrane components, and the factors that determine the characteristic fibrillar pattern of bound CSP. The fluorescence photobleach method (7-9) was used to measure rates of macroscopic lateral motion of fluorescently labeled CSP and antibodies to CSP and to assess the extent of interaction between CSP and other cell surface components, including unselected surface antigens, an exogenous fluorescent ganglioside analogue, a lipid probe, and concanavalin A (Con A) binding components. MATERIALS AND METHODSGoat antibodies against isolated, electrophoretically purified CSP were prepared and affinity-purified (5). Rhodaminelabeled antibodies were prepared by using tetramethylrhodamine isothiocyanate (7). CSP was isolated and purified as described (6). The protein was stored at a concentration of 1.6 mg/ml in 10 mM cyclohexylaminopropanesulfonic acid, pH 11/0. 15 M NaCl/I mM CaC12 in liquid nitrogen. CSP was labeled with fluorescein isothiocyan4te by dialyzing 1.1 mg of CSP (in 0.7 ml) for 24 hr against phosphate-buffered saline (Pi/NaCI) (Grand Island Biological) at 40, followed by dialysis against 20 ml of fluorescein isothiocyaanite (100 jg/ml) in 0.05 M bicarbonate-carbonate, pH 9.7/0.15 M NaCI overnight at 4°. To remove excess free dye, the CSP was dialyzed for 4 additional days against the bicarbonate-carbonate buffer at 40. Fluorescein-labeled CSP (F-CSP) was kept at 1 mg/ml in aliquots of 0.2-0.3 ml in liquid nitrogen.Primary and secondary CEF and 3T3 cells were grown in 35-mm plastic tissue culture dishes at 370 in a 95% air/5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium containing 5% (vol/vol) fetal calf serum. For labeling with F-CSP, the cells were incubated overnight with F-CSP ...

G protein-coupled receptor kinases and arrestin proteins are well-characterized mediators of agonist-dependent G protein-coupled receptor desensitization. These proteins are now shown to play a dual role in receptor regulation by mediating both receptor uncoupling and sequestration, a process important for receptor resensitization. b-Arrestins bound to phosporylated b2-adrenergic and angiotensin II type 1A receptors act as intracellular trafficking molecules specifically targeting these receptors for dynamin-dependent clathrin-coated vesicle-mediated sequestration.