Cell surface receptors for insulin and human growth hormone. Effect of microtubule and microfilament modifiers.

The receptors for the polypeptide hormones, insulin and growth hormone, are located on the cell surface. Since the cytoplasmic microtubules and microfilaments are involved in the mobility and distribution of surface receptors for immunoglobulins and lectins, we investigated the role of these structures in the binding of insulin and human growth hormone to their receptors on cultured human lymphocytes (IM-9). Cells preincubated with microfilament modifiers, cytochalasin A, B, and D (10 mug/ml), had decreased binding of insulin (30%) and human growth hormone (60%) under steady state conditions, which was not reversed by removing the cytochalasins from the medium and was due entirely to a reduced number of receptor sites on the cell surfact. The lost receptors were not detected in the medium, suggesting a redistribution within the cell. The cytochalasins failed to alter the affinity of the hormones for their receptors or the negative cooperativity of the insulin receptor. The anti-microtubule agents (vincristine, vinblastine, colchicine) had no effect on the binding of insulin and growth hormone to their receptors. Deuterium oxide, a stabilizer of microtubules and other proteins, decreased the affinity (40%) of insulin for its receptors under steady state conditions and accelerated moderately the spontaneous dissociation of 125I-insulin from its receptors. Since cytochalasin decreases the number of available insulin and human growth hormone receptor sites, cytochalasin-sensitive microfilamentous structures appear to modulate the exposure of cell surface hormone receptors, while microtubules do not seem to be involved.     

Microtubule inhibitors differentially affect translational movement,cell surface expression,and endocytosis of transferrin receptors in K562 cells

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleachig parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37°C and 21°C were calculated to be 0.10-0.15 min^?1 and 0.02–0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by β-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtuble inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells. © 1994 Wiley-Liss, Inc.     

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

The lymphocyte plasma membrane: locus of control in the immune response.

The lymphocyte plasma membrane is the locus of events which control the immune response. T and B lymphocytes, which mediate cellular and humoral immunity respectively, show distinctive plasma membrane morphologies and cell surface receptors. The dynamic state of these plasma components is emphasized by their lateral mobility in the fluid plane of the membrane, as well as variation in their structure or expression as the lymphocyte proliferates and differentiates in response to stimulation by antigen or mitogens. The best understood membrane glycoproteins are surface membrane immunoglobulins that serve as antigen receptors on B cells, and the histocompatability-beta2 microglobulin complex that has an immunoglobulin-like structure. Other less well defined surface structures showing modulation during the cell cycle may affect growth regulation of proliferating lymphocytes. Some of these are shared by fetal and neoplastic cells. Major theories of lymphocyte signaling are discussed, and the early events in lymphocyte activation are reviewed. While a complete model encompassing all these early events is not yet possible, the central issues can be usefully discussed in term of receptor-transducer-effector concepts derived by strong parallels from a knowledge of hormone-membrane interactions.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Transmembrane interactions and the mechanisms of transport of proteins across membranes

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin. Patching and capping of specific receptors on rounded cells is often accompanied by a specific endocytosis of the ligand-receptor complexes. This represents one common transport mechanism of a protein (the ligand) across the plasma membrane. The possibility is discussed that this type of endocytosis is mediated by a transmembrane linkage of the clustered receptor to actin/myosin. Another mechanism of endocytosis involves the “coated pit” structures that are observed by electron microscopy of plasma membranes. The possible relationships between an actin/myosin and a coated pit mechanism of endocytosis are explored.     

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.     

Kinetic evidence for a common mechanism of capping on lymphocytes

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for `fast' and `slow' cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or θ antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37°C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10μm-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10μm) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of θ antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not θ antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.     

Exocytotic "constipation" is a mechanism of tubulin/lysosomal interaction in colchicine myopathy

Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis. Thus, we examined, as a possible cellular pathogenesis of colchicine myopathy, how the muscle cytoskeleton affects the degradation of membrane proteins, which are processed through the endosomal/lysosomal pathway. We used the acetylcholine receptor as a model membrane component in cultured myotubes allowed to preincubate with colchicine. We tested at which step colchicine interferes with receptor trafficking by accounting for internalization, delivery to lysosomes, hydrolysis, or exocytotic release of debris. We report that colchicine significantly decreases the exocytosis of AChRs but does not affect receptor internalization, lysosomal hydrolysis, or the number of surface membrane receptors. Further, our immunofluorescence observations revealed a morphologic tubulin network in rat skeletal muscle that is more densely distributed in white (mitochondria-poor) muscle fibers than in red (mitochondria-rich) fibers but is present in both. Ultrastructurally, immunogold labeling localized tubulin in the intermyofibrillar region in a long and linear fashion, unassociated with myofibers or mitochondria. Taken together, our findings suggest the following: (1) Microtubules likely play a functional role in the pathway of lysosomal degradation in normal adult skeletal muscle; (2) The observed decrease in overall apparent degradation of membrane receptors by colchicine must be due primarily to inhibition of exocytosis. These data indicate that lysosomal "constipation" underlies colchicine myopathy. (3) An animal model faithful to the human disorder will allow further pathogenetic studies.     

Binding of purified lectins to guinea pig lymphocytes. Studies of the number, binding constant, and distribution of lens culinaris lectin A and Agaricus bisporus lectin molecules on lymphocyte surfaces

The surface membrane of guinea pig lymphocytes contains discrete receptors for several different homogeneous lectins. Two very similar lectins from the lentil, LcL-A and LcL-B, bind to the same receptor. Three other lectins, AbL, WGA, and Con A, do not bind detectably to the LcL receptor. AbL binds to another discrete receptor to which none of the other lectin molecules bind. The LcL and AbL receptors are contained on different units, each of which is capable of independent movement on the cell surface. The normal distribution of LcL molecules on the surface is different than the distribution of AbL molecules. However, when lymphocytes are simultaneously incubated with LcL and AbL, the distribution of bound LcL molecules on the cell surface parallels the normal distribution of AbL molecules, which indicates that the movement of AbL molecules and AbL receptors on the surface can strongly influence the movement of LcL and LcL receptors. When lymphocytes are held in close contact by LcL or AbL, the lectin molecules and receptors appear to concentrate at the area of contact between two cells. A model is presented which suggests that the migration of lectins and lectin receptors can occur by a multicellular process.     

Observations on transmembrane structures of surface immunoglobulin in the plasma membrane of B lymphocytes

We have investigated the possible role of intramembraneous particles as revealed by freeze-fracture electron microscopy in the plasma membrane of B lymphocytes from rabbits and mice as reflections of transmembrane structures of surface immunoglobulin receptor molecules. This was achieved by aggregation of the surface receptors using fluorochrome-conjugated antibodies, fixation and freezing of the cells in 35% glycerol. This procedure resulted in replicas of lymphocytes with well-preserved morphology (no ice-crystals), enabling the study of both protoplasmic and external fracture face in combination with surface receptor markers. It appeared that very small intramembraneous particles (3–6 nm diameter) were selectively clustered under patches of surface receptor label. This phenomenon was found on the external fracture face exclusively and not on the protoplasmic fracture face. ‘Classical’ intramembraneous particles (6–12 nm diameter) were not involved. We suggest that these small, clustered particles should be interpreted as transmembrane structures of surface immunoglobulin molecules.     

Single Particle Tracking reveals two distinct environments for CD4 receptors at the surface of living T lymphocytes

We investigated the lateral diffusion of the HIV receptor CD4 at the surface of T lymphocytes at 20°C and 37°C by Single Particle Tracking using Quantum Dots. We found that the receptors presented two major distinct behaviors that were not equally affected by temperature changes. About half of the receptors showed a random diffusion with a diffusion coefficient increasing upon raising the temperature. The other half of the receptors was permanently or transiently confined with unchanged dynamics on raising the temperature. These observations suggest that two distinct subpopulations of CD4 receptors with different environments are present at the surface of living T lymphocytes.     

Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Isolation of the receptors by antiligand-affinity chromatography

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.     

The effect of amphotericin B on lectin-induced aggregation of cell surface receptors

The mobility of concanavalin A (ConA) and ricin receptors from NS20 neuroblastoma and C₆ glioma cells was studied using an electrophoretic technique. Cells attached to a solid support were exposed to an electrical field (12V cm⁻¹) at room temperature. The distribution of lectin receptors on the cell surface was revealed by fluorescent conjugates of lectins and microscopic observation of the fixed cells. This technique allowed the estimation of the mobilities of lectin receptors either in free or liganded form, depending on the time at which the cells are labeled with lectins (either after or before electrophoresis). In line with previous observations [1] it is shown that in their free form ConA and ricin receptors are mobile all over the cell surface. Ligand binding induced an apparent receptor immobilization. Immobilization of ricin receptors from C₆ glioma cells could be induced either by the multivalent or the monovalent form of the lectin indicating that cross-linking of receptors by the ligand did not play a predominant role in the process of receptor immobilization. Amphotericin B but not ionophores like valinomycin or gramicidin blocked ligand-induced receptor immobilization. It is concluded from this observation that the effect of amphotericin B is not related to its ionophoretic properties but more likely to its capacity to interact with membrane cholesterol. When cells were incubated at 37 °C extensive patching of lectin receptors could be observed. This process was also inhibited by amphotericin B. A model is proposed to account for a role of cholesterol in ligand-induced receptor immobilization and patching.     

The lymphocyte plasma membrane: Locus of control in the immune response

Summary The lymphocyte plasma membrane is the locus of events which control the immune response. T and B lymphocytes, which mediate cellular and humoral immunity respectively, show distinctive plasma membrane morphologies and cell surface receptors. The dynamic state of these plasma membrane components is emphasized by their lateral mobility in the fluid plane of the membrane, as well as variation in their structure or expression as the lymphocyte proliferates and differentiates in response to stimulation by antigen or mitogens. The best understood membrane glycoproteins are surface membrane immunoglobulins that serve as antigen receptors on B cells, and the histocompatability-β ₂ microglobulin complex that has an immunoglobulin-like structure. Other less well defined surface structures showing modulation during the cell cycle may affect growth regulation of proliferating lymphocytes. Some of these are shared by fetal and neoplastic cells. Major theories of lymphocyte signaling are discussed, and the early events in lymphocyte activation are reviewed. While a complete model encompassing all these early events is not yet possible, the central issues can be usefully discussed in terms of receptor-transducer-effector concepts derived by strong parallels from a knowledge of hormone-membrane interactions. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975. Supported by: Medical Research Council of Canada and National Cancer Institute of Canada. Scholar of the Medical Research Council of Canada.     

Ligand-selective targeting of the glucocorticoid receptor to nuclear subdomains is associated with decreased receptor mobility

The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.     

Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs,monensin, low temperature,and chloroquine

We studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for ¹²⁵I-asialo-orosomucoid binding, to measure receptor activity, or ¹²⁵I-anti-Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin-permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub-37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ? 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin-and chloroquine-treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.     

Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation.     

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.