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Spontaneous redistribution of cell-surface glycoproteins in lymphoid cells during cytokinesis.

The 'unperturbed' distribution of plasma membrane glycoproteins during cytokinesis has been examined by immunofluorescence and electron microscopy on dividing mouse and rat lymphoid cells fixed before being labelled with the appropriate reagents. Two groups of molecules which cap 'spontaneously' to the uropod of non-dividing cells, i.e., the common receptors for Helix pomatia (HPA) and peanut agglutinin (PNA) (and in particular the thymocyte glycophorin-like glycoprotein) and membrane immunoglobulins, redistribute spontaneously to the cleavage furrow during cytokinesis. By electron microscopy, the redistributed molecules (HPA receptors) appear to be aggregated in clusters. Other glycoproteins, such as Concanavalin A receptors and Thy.1 antigens, which do not cap spontaneously on interphase cells, remain uniformly distributed or are somewhat depleted over the cleavage furrow. The results suggest that a spontaneous 'transport' of certain membrane molecules from the nuclear pole to the cleavage furrow occurs normally during cytokinesis by a mechanism analogous to that of uropod formation and spontaneous capping in interphase cells. The existence of redistribution phenomena in dividing cells imposes some restrictions on the possible mechanisms of redistribution and on certain aspects of the cleavage process.

Animals↗

Increased stability of E-rosettes and restricted capping of sheep erythrocytes by lymphocytes of aged humans.

The processes of E-rosette dissociation and sheep red blood cell (SRBC) capping provide simple assays for studying age-related changes in membrane dynamics of T-lymphocytes. After incubation at 4 degrees C, no significant difference is observed between young-adult and elderly subjects, either in the number of rosette-forming-cells (E-RFC) or in the distribution of SRBC at the lymphocyte surface. However, when the E-RFC are incubated at 22 or 37 degrees C after resuspension, the rosettes disintegrate to a larger extent forming fewer morula-like structures and more caps in young donors. An inverse relationship is noted between the number of E-RFC and the percentage of capping cells, suggesting a role for the lateral movement of the SRBC receptors in the dissociation process. In the elderly, rosette disintegration seems to be related only to the random release of SRBC. It is speculated that this increased stability of the E-rosettes represents some locking of the T-lymphocyte membrane receptors, which could alter the transduction of cell-cell signals.

Adult↗

Surface immunoglobulin, lectin-induced cap formation, and phagocytic function in five patients with the leukemic phase of hairy cell leukemia.

Hairy cells from 5 patients with greater than 25% hairy cells in the peripheral blood (4 had greater than 45% hairy cells and white blood cell counts (WBC) greater than 10,000/mm3) were studied for surface immunoglobulin (SIg) presence and distribution by two methods, for lectin-induced cap formation, and for phagocytosis of zymosan. Hairy cells from all 5 cases were found to have distinct monoclonal patterns, 2 Gk, 1 MDk, 1 Dk, and 1 GMDk, as well as cap formation with SIg. All 5 cases showed distinct lectin-induced cap formation in a percentage of cells similar to the percentage of hairy cells, and 2 of the 5 patients had hairy cells which phagocytosed zymosan. These findings contrasted with the malignant cells from 4 patients with CLL, which had monoclonal SIg but no SIg cap formation and no significant percentage of lectin-induced cap formation. Cells from 2 cases of T cell lymphomas had no SIg and no lectin-induced cap formation as did cells from 2 cases of non-lymphocytic leukemia. Hairy cells not only appear to have SIg cap formation similar to some B-lymphocytes, which in some patients also phagocytose zymosan, but also demonstrate strong lectin-induced cap formation.

Adult↗

Effect of anti-T cell autoantibodies from systemic lupus erythematosus sera upon T lymphocyte functions.

This study was undertaken to establish whether IgM and IgG anti-T cell autoantibodies obtained from sera of patients with active systemic lupus erythematosus, impair normal T lymphocyte functions. Two in vitro models of T cell function were examined: (a) the capacity of cells to cap, endocytose, and regenerate the T3, T4, and T8 surface antigens; and (b) the adenosine-induced T4----T8 phenotype switch. The results demonstrated that autoantibody neither impaired the capping process, nor impeded the phenotypic switch. Thus, bound anti-T cell autoantibodies do not appear to interfere with these specific T lymphocyte functions and cannot directly account for either the impaired T cell capping mechanism or the block in adenosine-induced phenotype switch observed during active systemic lupus erythematosus.

Autoantibodies↗

Linkage between surface immunoglobulin and cytoskeleton of B lymphocytes may involve Gc protein.

The membrane immunoglobulin (MIg) of B lymphocytes is thought to have an important role in antigen recognition and cellular activation. In common with many membrane glycoproteins, MIg moves extensively in the lipid bilayer, and after binding of specific antisera displays lateral mobility with patch and cap formation. This phenomenon appears to involve the cytoskeleton, particularly the actin that is present in the cell membrane of B lymphocytes and aggregates beneath capped immunoglobulin. Recently, it has been reported that the isolation of MIg results in co-purification of actin and an unknown protein of molecular weight (MW) approximately 56,000 (refs 7, 8). We now demonstrate that this component displays physicochemical and immunological properties indistinguishable from those of Gc (group-specific component). In addition, evidence is presented which suggests that this vitamin D3-binding protein is involved in the linkage between MIg and actin, and may therefore be important in signal transduction.

Actins↗

Effect of concanavalin A dose, unbound concanavalin A, temperature, Ca2+ and Mg2+, and vinblastine on capping of concanavalin A receptors of human peripheral blood lymphocytes.

The capping of Concanavalin A (Con A) receptors induced by Con A was studied using human peripheral blood lymphocytes. The effects of Con A dose (5--100 micrograms/ml), pretreatment at 4 degrees C, unbound Con A, extracellular Ca2+ and Mg2+ and vinblastine were evaluated using Con A-horseradish peroxidase and electron microscopy. Lymphocytes incubated with Con A at 4 degrees C and fixed with glutaraldehyde exhibited Con A-horseradish peroxidase around the entire cell periphery. After raising the temperature to 37 degrees C, the Con A-horseradish peroxidase moved to form a cap at one pole of the cell and subsequently underwent endocytosis. Capping of Con A receptors induced by Con A at 37 degrees C was observed only at low Con A concentrations in the presence of unbound Con A and extracellular Ca2+ and Mg2+. Increased capping was found after pretreatment of cells with Con A at 4 degrees C, removing unbound Con A and/or removing extracellular Ca2+ and Mg2+, and by treatment with vinblastine. Following removal of both unbound Con A and extracellular Ca2+ and Mg2+, the percentage of capped cells at 37 degrees C was the same as on pretreatment at 4 degrees C under the same conditions. While pretreatment at 4 degrees C caused the breakdown of microtubules, removal of unbound Con A and/or extracellular Ca2+ and Mg2+ had no morphological effect on microtubules or microfilaments. Following exposure of lymphocytes to vinblastine and removal of unbound Con A, capping of Con A receptors by Con A was observed in over 90% of cells at all Con A dosages. However, when cells were exposed to vinblastine in the presence of unbound Con A the formation of Con A caps was either partially or completely inhibited.

Calcium↗

Role of cholesterol in the capping of surface immunoglobulin receptors on murine lymphocytes.

Previously, we have shown that the capping of surface immunoglobulins on murine lymphocytes can be affected by modulating the lipid environment of the surface membrane with free fatty acids. In the present study, murine lymphocytes were depleted of cholesterol by incubation with phospholipid vesicles. As the cellular cholesterol:phospholipid ratio decreased, the capping of the surface immunoglobulin was seen to decrease. This inhibition of capping could not be reversed by calcium and is not accompanied by changes in either the cytoskeletal element alpha-actinin or cellular ATP levels. Incubation of the cholesterol-depleted cells with cholesterol-containing phospholipid vesicles raised both the cholesterol:phospholipid ratio and capping levels to values close to those of untreated control cells. Remarkably, stearic acid, a saturated fatty acid, could also restore the capping levels in the cholesterol-depleted cells. On the basis of the present data and measurements of the fluorescence polarization of the probe diphenyl hexatriene, we propose a model in which the protein(s) involved in capping is located in a gel-like lipid domain, and that removal of cholesterol makes this domain less gel-like and inhibits capping. Restoration of the gel-like nature of this domain by the addition of either cholesterol or stearic acid enables the protein(s) to function normally.

Actinin↗

Translocations of fodrin and its binding proteins.

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I: two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.

Animals↗

Mobility of the human T lymphocyte surface molecules CD3, CD4, and CD8: regulation by a cAMP-dependent pathway.

The present study was undertaken to determine whether a cAMP pathway mediates the mobility of CD3, CD4, and CD8 within the membrane. Crosslinking CD3, CD4, and CD8 with monoclonal antibody and anti-antibody induced rapid accumulation of intracellular cAMP, occupancy of cAMP receptors, and was temporally associated with the mobilization and directed movement of these molecules to a pole of the cell. This capping process could be partially inhibited in a dose-dependent manner by treatment of T cells with 2',5'-dideoxyadenosine, a ribose-modified adenosine analogue that binds to the P site of the catalytic subunit of adenylate cyclase and reduces adenylate cyclase activity. Furthermore, inhibition of cAMP-dependent endogenous phosphorylation of 17.5-kDa, 23/25-kDa, and 33.5-kDa bands in intact T cells by N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide, a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, blocked the capping event. Data support the conclusion that crosslinking of CD3, CD4, and CD8 activates a cAMP-dependent pathway that mediates the mobilization and directed movement of these molecules. cAMP-dependent protein phosphorylation is an integral step leading to the capping process.

Adenylyl Cyclase Inhibitors↗

Immunization of mice with syngeneic Moloney lymphoma cells induces separate antibodies against virion envelope glycoprotein and virus-induced cell surface antigens.

Immunization of mice with heavily irradiated syngeneic Moloney lymphoma cells evokes antibodies against the major viral envelope antigen, gp71, and the Moloney virus-induced cell surface antigen (MCSA). A9HT cells, an L-cell subline, react with the antibodies against the viral envelope antigen only; this reaction can be completely inhibited by virus or purified gp71. Reactivity to Moloney lymphoma cells (YAC) was only partially inhibited (maximum 30%) or not at all. This can be attributed to the reaction of the YAC cells with antibodies directed against MCSA, a nonvirion cell surface component according to both biological and biochemical evidence. Antibody-induced capping of gp71 or p15(E) did not change the membrane distribution of MCSA or H-2, indicating that these antigens represent distinct entities on the cell surface. MCSA showed only minimal capping and thereby differed in behavior from both H-2 and virion antigens. gp71 could be capped by the mouse antiserum as revealed by subsequent staining with monospecific anti-gp71 antiserum. Under ordinary test conditions this reactivity is overshadowed by the reaction against MCSA. The lack of MCSA capping reflects a difference in anchorage of this antigen.

Antibodies, Viral↗

Diminished capping of lymphocytes from pregnant women.

The capping of lymphocytes from normal pregnant women is diminished. Decreased capping is observed for both anti-Ig and for Con A receptors. The altered capping response appears between the tenth and the twelfth week of pregnancy. The diminution in capping persists until about 2 weeks after delivery. The possible association between the decreased capping response and the altered immune responses known to occur during pregnancy is discussed. The factors underlying the altered capping response in pregnancy may be related to the factors responsible for the parallel phenomenon in pathological conditions.

Antibodies, Anti-Idiotypic↗

Lectin-bearing protoplasts of Pseudomonas aeruginosa induce capping in human peripheral blood lymphocytes.

The redistribution of surface membrane receptors and cap formation by pseudomonas aeruginosa lectins (Ps-GAL, which binds D-galactose, and Ps-MAN, which binds D-mannose and L-fucose) was studied in human peripheral blood lymphocytes by scanning electron microscopy. When Ps-GAL and Ps-MAN-bearing protoplasts of Pseudomonas aeruginosa were incubated with lymphocytes, cap formation was revealed by the accumulation of protoplasts at one pole of the lymphocyte. No adherence or capping was observed when intact Pseudomonas aeruginosa cells were used. Pretreatment of the lymphocytes by papain or neuraminidase did not enhance the capping. No capping or adherence of protoplasts to lymphocytes was observed in the presence of D-galactose, while addition of D-mannose had no effect. The inhibitory effect of D-galactose on the adherence and capping in human peripheral lymphocytes by Pseudomonas aeruginosa protoplasts suggests that the Ps-GAL lectin is responsible for both phenomena.

Galectins↗

Capping of complement receptors on human neutrophils induced by group A streptococcal cell walls.

Peptidoglycan-polysaccharide polymers derived from group A streptococcal cell walls (PG-PS) were opsonized with either purified C3 or normal human serum and were used as a probe to investigate the mobility of CR1 and CR3, the C3b and iC3b receptors, respectively, on human neutrophils. Incubation of monolayers or cell suspensions of neutrophils with PG-PS opsonized with C3b or serum resulted in capping of PG-PS, as detected by fluorescein-labeled antibody to PS. No binding of PG-PS to neutrophils was observed with heat-inactivated serum. By 30 min the cell walls were internalized and observed in one to three vacuoles. Capping was totally inhibited when PG-PS opsonized with C3b or serum was preincubated with Fab'-anti-C3b. Similar inhibition was observed when C3b-opsonized PG-PS was incubated with neutrophils that were preincubated with anti-CR1 or fluid-phase C3b; only partial inhibition of neutrophil capping was observed by using serum-opsonized PG-PS. Because anti-CR1 blocks only the C3b receptor, the cap formation observed with serum-opsonized PG-PS is probably due to CR3. These results suggest that both CR1 and CR3 on neutrophils cap after stimulation by group A streptococcal cell wall fragments.

Animals↗

Effect of the antidepressant drug-Stelazine on lymphocyte capping.

The antidepressant agent, Stelazine (trifluoroperazine dihydrochloride), is known to cause inactivation of the calcium-binding protein, calmodulin. We report here that this drug in the concentration range of 5 x 10(-5) M to 10(-6) M causes remarkable inhibitory effets on ligand-induced T-lymphocyte receptor capping. The possible involvement of calmodulin in the regulation of lymphocyte surface receptor movement is therefore implicated.

Animals↗

Concanavalin A receptor capping in mouse myeloma and hybridoma.

Concanavalin A capping was studied in immunoglobulin-secreting hybridomas derived from fusion of mouse myeloma NSO cells with mouse spleen lymphocytes. The cells of the parental populations differed significantly in capping ability (low in myeloma cells and high in the lymphocytes). Among the hybridoma cells tested, several clones showed low capping, similar to that of the myeloma cells, some showed a good degree of capping, similar to that of the lymphocytes and other clones expressed an intermediate capping response. Capping was significantly increased in hybridoma clones of intermediate capping ability following in vivo intraperitoneal growth. A possible relationship of the variation in capping response to cell motility and to metastatic capacity is pointed out.

Animals↗

Cross-linked surface Ig attaches to actin.

In lymphocytes and P3 myeloma cells, cross-linking of surface Ig by the capping and patching phenomenon has been used to demonstrate the induction of a specific association between surface Ig and cellular actin.

Actins↗

Duration of the calcium signal in the mitogenic stimulation of thymocytes.

An increase in the free cytoplasmic Ca2+ concentration in thymocytes can be detected by the fluorescent indicator quin 2 within a few seconds of the addition of concanavalin A and the response is quantified from the increased proportion of quin 2 in the cells chelated by Ca2+ ('% Ca-quin 2'). The % Ca-quin 2 in untreated cells is 53 +/- 6%, increases to 64 +/- 7% immediately after the addition of concanavalin A and declines spontaneously over 24 h back to the level in untreated cells (53 +/- 6%). The increase in % Ca-quin 2 in response to concanavalin A is completely blocked when 50 mM-alpha-methyl D-mannoside is added before concanavalin A and completely reversed when the competing sugar is added immediately after the mitogen. Addition of alpha-methyl D-mannoside at increasing intervals after concanavalin A addition causes a progressively smaller decrease in % Ca-quin 2 and has a negligible effect after 24 h, when the % Ca-quin 2 is the same as that in untreated cells. The decline in the calcium signal defined by these experiments has a similar time course to cap formation by concanavalin A on the cells. It is concluded that the calcium signal lasts only while concanavalin A is bound to the cell surface and is terminated either by capping or by the addition of alpha-methyl D-mannoside.

Aminoquinolines↗