Ascorbic acid and catecholamine release from digitonin-treated chromaffin cells

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.     

Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin

The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular 86Rb+ and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.     

Rotational motion of monomeric and dimeric immunoglobulin E-receptor complexes.

Erythrosin 5'-thiosemicarbazide labeled immunoglobulin E (IgE) was used to monitor the rotational dynamics of monomeric and dimeric Fc epsilon RI receptors for IgE on rat basophilic leukemia (RBL) basophilic leukemia (RBL) cells using time-resolved phosphorescence anisotropy. Receptors were studied both on living RBL cells and on membrane vesicles derived from RBL cell plasma membrane. The un-cross-linked IgE-receptor complexes on cells and vesicles exhibit rotational correlation times that are consistent with those expected for freely rotating monomers, but a small fraction of these complexes on cells may be rotationally immobile. A comparison of the initial phosphorescence anisotropy values for erythrosin-labeled IgE-receptor complexes on cells and vesicles reveals a fast component of rotational motion that is greater on the vesicles and may be due to a site of segmental flexibility in the receptor itself. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibodies appear to be largely immobile on cells, but they are mobile on vesicles with a 2-fold larger rotational correlation time than the monomeric complexes. The results suggest that dimeric IgE-receptor complexes undergo interactions with other membrane components on intact cells that do not occur on the membrane vesicles. The possible significance of these interactions to receptor function is discussed.     

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Pre-treating donor cells before somatic cell nuclear transfer (SCNT, 'cloning') may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.     

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.     

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.     

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell- cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.     

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol cholesterol content of resistant cells and recovery of streptolysin O sensitivity

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20α-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, ²³Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na⁺ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

Maintenance of motility in mouse sperm permeabilized with streptolysin O

One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [gamma-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.     

Protein phosphorylation in permeabilized pancreatic islet cells.

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.     

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589- 4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.     

Real-time analysis of membrane permeabilizing effects of oleanane saponins

Saponins are a group of plant and marine derived glycosides with numerous biological functions. Two important characteristics of certain plant saponins are their ability to enhance cytotoxicity of type I ribosome inactivating proteins and stimulation of the immune system. The main objective of the present study was to investigate in real-time the permeabilizing effects of saponins on cell membrane. A set of oleanane saponins (glycyrrhizinic acid, Gypsophila, Saponaria and Quillaja saponins) and a steroid saponin (digitonin) were tested. The effects of these saponins on lysosomal membranes and hemolysis, along with their charge were also studied. Real-time monitoring of cell membrane permeabilization facilitated a highly sensitive analysis of the cellular kinetics. Saponins showed variable permeabilizing effects on cellular and lysosomal membranes at concentrations from 6 μM and hemolysis from 3 μM. Further, the results suggest that charge of the saponin may be relevant for permeabilizing effects of oleanane saponins.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, 23Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na+ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.     

Bcl-2 changes conformation to inhibit Bax oligomerization

Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.     

两种人胃癌细胞M期与间期时相质膜表面Con A受体复合物分布及侧向运动

利用荧光标记和荧光漂白恢复方法研究了两种人胃腺癌细胞M期与间期时梠中,细胞膜表面ConA受体复合物分子的分布与侧向运动.结果表明:MGC80-3细胞M期时相与SGC7901细胞间期时相膜表面ConA受体复合物分布近似,其侧向运动方式呈扩散型;SGC 7901 M期时相细胞膜表面ConA受体复合物的分布与MGC80-3细胞间期时和相基本类似,其侧向运动主要是流动型.凡是受体复合物是流动型运动的细胞.其膜上可动分子的百分比都寓于扩散型运为的细胞,P值小于0.01.     

Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells

The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coefficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 X 10(-10) cm2/S less than or equal to D less than or equal to 4 X 10(-10) cm2/S) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 +/- 19% at 25 degrees C (82 +/- 14% at 37 degrees C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 +/- 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 +/- 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum.