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Biomedical subjects

J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 379 records · Page 21Linked to original sources
PubMed1980

Lateral diffusion of membrane lipids and proteins during the cell cycle of neuroblastoma cells.

The fluorescence photobleaching recovery method has been used to determine the lateral mobilities of membrane lipids and proteins during the cell cycle of synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A). As probes for lipid mobility, 3,3'-dioctadecylindocarbocyanine iodide and a fluorescein-labeled analog of ganglioside GM1 were used. Membrane proteins were labeled with rhodamine-labeled rabbit antibodies against mouse E14 cells. For both lipid probes the diffusion coefficients reach a minimum in mitosis, increase 2- to 3-fold during G1, remain constant at maximal values during S, and decrease again shortly before mitosis. Membrane proteins also exhibit minimum diffusion coefficients in mitosis, followed by a similar rise in G1. However, as cells proceed through S and G2, the lateral mobility of the membrane proteins gradually decreases. It is argued that lipid mobility is controlled by the fluidity of the membrane lipid matrix whereas protein mobility is governed also by other constraints.

Animals↗
PubMed1980

Mobility, clustering, and transport of nerve growth factor in embryonal sensory cells and in a sympathetic neuronal cell line.

We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. We have used this analogue to study the mobility and distribution of NGF receptors on embryonal chicken sensory cells from dorsal root ganglia and on a pheochromocytoma cell line (PC-12) that responds to NGF by differentiating along a neuronal pathway. The rhodamine conjugate of nerve growth factor (R-NGF) binds initially to diffusely distributed mobile receptors (D approximately 8 X 10(-10) cm2/sec) on immature sensory and PC-12 cells. At 37 degrees C, the NGF receptor complexes cluster and form immobile visible patches. These patches undergo endocytosis in a process that consumes metabolic energy. Methylamine blocks the formation of visible patches of NGF and the receptors remain dispersed and mobile at 37 degrees C. On differentiated chicken sensory cells, R-NGF binds to diffusely distributed mobile receptors and to aggregated immobile binding sites. These clusters are localized at the tip of the axon, along the axon, and in the main body. The NGF molecules that are internalized at the tip of the axon are transported retrogradely from the peripherey to the cell body.

Animals↗
PubMed1979

Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells.

Lateral diffusion of membrane lipids and proteins was determined in differentiating C1300 mouse neuroblastoma cells by fluorescence photo-bleaching recovery measurements. It is demonstrated that upon differentiation the lateral diffusion of membrane lipids and proteins is increased specifically in the extending neurites. This indicates the appearance of a topographical heterogeneity in the cell membrane, whereby more fluid domains become located in the membrane of the neurites.

Animals↗
PubMed1979

Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor.

An analogue of epidermal growth factor (EGF) which is virtually devoid of biological activity retains receptor binding activity but cannot form cell surface clusters or patches. Bivalent anti-EGF antibodies restore both bioactivity and patch formation. The sensitivity of fibroblasts to native EGF can also be enhanced greatly by these antibodies, especially in hormone-resistant cell lines.

Animals↗
PubMed1979

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.

Animals↗
PubMed1978

Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization.

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.

Cell Line↗
PubMed1978

Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts.

Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D approximately (3--5) x 10(-10) cm2/sec at 23 degrees C. Increasing the temperature to 37 degrees C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.

Azides↗
PubMed1978

Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

Adipose Tissue↗
PubMed1978

Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells.

We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 fibroblasts. We have used two types of fluorescent analogues of insulin and epidermal growth factor: highly fluorescent derivatives which have seven to eight rhodamine molecules or fluorescent derivatives which have a single rhodamine molecule per one molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogues were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4 degrees , remained dispersed. Within a few minutes at 23 degrees or 37 degrees the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initially mobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within approximately 30 min at 37 degrees , much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.

Cell Line↗
PubMed1977

Lateral transport of a lipid probe and labeled proteins on a cell membrane.

Diffusion coefficients (D) of a lipid probe and labeled proteins on L-6 myoblast membranes have been measured giving D(protein) approximately 2 X 10(-10) square centimeter per second and D (lipid probe) approximately 9 X 10(-9) square centimeter per second. Some of the membrane proteins are immobile, but the lipid probe diffuses freely over macroscopic distances. Cytochalasin B slows protein but not lipid probe diffusion.

Azides↗
PubMed1977

Receptor diffusion on cell surfaces modulated by locally bound concanavalin A.

In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.

Antigen-Antibody Complex↗
PubMed1977

Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components.

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 bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.

Antigens↗