Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.     

Relationship between phosphatidylcholine biosynthesis and cellular commitment in concanavalin A-stimulated lymphocytes

The incorporation of [¹⁴C]choline into phosphatidylcholine was studied in lymphocyte cultures exposed to concanavalin A (ConA). The lectin was found to induce an increase in the incorporation of the label with following features:

1. 1. It occurs very promptly after exposure.

2. 2. It is not elicited by a non-mitogenic lectin.

3. 3. The increase in the early stage is proportional to lectin concentration.

4. 4. It can be terminated by a competitive inhibitor of ConA binding.

5. 5. The extent of the increase shows a correlation with the rate of cellular commitment to initiate DNA synthesis. These results suggest that in the mitogenic stimulation of T lymphocytes enhanced synthesis of membrane phospholipids is a precommitment event.

     

Immobilization of lymphocytes at surfaces by lectins

Phytohemagglutinin (PHA) and Concanavalin A (ConA) cause normal chicken lymphocytes to adhere to glass and plastic surfaces. Pokeweed mitogen (PWM) does not cause adherence. The effect of ConA was studied in detail. The reaction begins within 15 min at 25 °C and proceeds to completion by 2 h. It is independent of pH and resembles in this respect the spontaneous adherence which can occur in protein-depleted suspensions of chicken lymphocytes. It is distinguished from spontaneous adherence by conducting the reaction in 1% or more serum protein; high concentrations exert a slight restraint, which can be overcome by increasing the concentration of ConA. The reaction is slightly greater at high cell concentrations, is inhibited by 3 mM sodium cyanide, and is effectively blocked by 3 mM iodoacetamide and the α-methyl- -glycosides of glucose and mannose. The reaction is not affected by 2-deoxy- -glucose or N-acetyl glucosamine. Adherent lymphocytes detach when the lectin solution is replaced with lectin-free saline; they readhere when reexposed to ConA or to alloantibody directed against lymphocyte surface antigen. At low concentrations of ConA the large lymphocytes of the bursa of Fabricius adhere more rapidly than the small lymphocytes of the blood and thymus. Mouse lymph node lymphocytes adhere in the same manner as small chicken lymphocytes.     

Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Cell surface changes in transformed human lymphocytes : I. ConA and E-PHA induced unique changes in surface topography

Binding studies with six purified plant lectins were used to investigate membrane alterations that occur in lymphocyte transformation. Normal human peripheral blood lymphocytes transformed with E-Phytohemagglutinin (E-PHA) or concanavalin-A (Con-A) generally possessed increased numbers of lectin receptors. When this increase was corrected for the expanded surface area of transformed lymphocytes, it appeared that E-PHA and ConA each produced a unique and complex reorganization of cell surface topography. Surface alterations occurred independently of DNA synthesis, cell proliferation, and microtubule or microfilament function. Puromycin inhibited emergence of new lectin receptors on cells transformed with E-PHA, but not with ConA. Lymphocytes incubated with either lectin showed increased incorporation of [¹⁴C]galactose into trypsin-sensitive cell surface glycoproteins. This incorporation was abolished by puromycin in cells stimulated by E-PHA but not by ConA. These studies demonstrate that although both lectins induce similar morphological alterations in human lymphocytes, at the molecular level the structural changes induced in the cell membrane by these two lectins differ considerably. Furthermore, these structural alterations are mediated via different mechanisms in the two groups of cells. De novo protein synthesis is required for cell surface reorganization in PHA-stimulated cells, but not in cells stimulated by ConA. The effect of ConA appears to be to enhance attachment of saccharide structures to pre-synthesized membrane proteins.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

Inhibition of concanavalin A-induced agglutination of Novikoff tumor cells by cytochalasins and metabolic inhibitors : Role of cell-surface morphology and the distribution of concanavalin A receptors

Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN₃ and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that

1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively.

2. 2. NaN₃ and DNP both inhibit cytoagglutination, but DNP is more effective and specific.

3. 3. Combinations of metabolic inhibitors (NaN₃ or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone.

4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN₃, DNP, or combinations of these drugs.

5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling.

6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not.

7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination.

These results suggest that the cytochalasins inhibit ConA-induced agglutination of Novikoff cells by their effect on cell-surface morphology, rather than by their effects on the topographical distribution of cell-surface lectin receptors, and that metabolic inhibitors reduce agglutinability by a different mechanism than the cytochalasins.     

Syndecan-4 is expressed by B lineage lymphocytes and can transmit a signal for formation of dendritic processes

Our previous studies indicated that stromal cell-derived syndecan-4 might mediate some form of communication with pre-B cells in bone marrow. We now report additional aspects of this recognition and show that syndecan-4 is also present on pre-B cells. Indeed, the molecule is acquired at an early stage of differentiation and retained until mature B cells undergo Ig isotype switching. mAbs developed to two portions of the syndecan-4 protein core were used to probe possible functions on B lineage lymphocytes. Syndecan-4 ligation had no obvious influence on B lymphocyte formation or activation, but this treatment caused a dramatic morphological change in appropriately stimulated leukocytes. Extended filopodia appeared on transfected Ba/F3 or FDCP-1 cells, as well as activated B cell blasts that were placed on syndecan-4 Ab-coated surfaces. The dendritic processes contained polymerized actin as well as pp52(LSP1), a prominent F-actin binding protein in lymphocytes. The cytoplasmic domain of syndecan-4 was not required for this response. Shape changes of this type could facilitate interactions between B lymphocytes and other components of the immune system. Not only is syndecan-4 a useful marker for discriminating normal B lineage lymphocyte subsets, but our results suggest new ways for the syndecans to participate in immune responses.     

A ground-based model to study the effects of weightlessness on lymphocytes

The mitogenic response of human lymphocytes was found to be markedly reduced in weightlessness conditions as compared to normal gravity. One possible explanation is that due to the non-existent sedimentation in space the lymphocytes could not adhere and spread on a substratum. Thus, we investigated the effect of substratum adhesiveness on lymphocyte responsiveness by reducing and blocking cell adhesion with poly-HEMA in a simple on-ground system. Lymphocyte adhesiveness was assessed by measuring the proportion of non-adhesive, slightly, and strongly adhesive 51Cr-radiolabelled cells on uncoated and poly-HEMA coated plastic. The amount of cell spreading on surfaces with varying adhesiveness was determined by measuring the area of cells. Cells grown on medium and thick poly-HEMA films were rounded in shape. By contrast, on tissue culture plastic, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.     

Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system

EGF-like sequences, inherent in a number of extracellular matrix proteins, participate in cell adhesion. It is possible that interactions of these sequences with EGF receptors (EGFR) affect actin filament organization. It was shown previously [Khrebtukova et al., 1991: Exp. Cell Res. 194:48-55] that antibodies specific to EGFR induce capping of these receptors and redistribution of cytoskeletal proteins in A-431 cells. Here we report that A-431 cells attach and spread on solid substrata coated with antibodies to EGFR, even in the absence of serum. Thus, EGFR can act as an adhesion protein and promote microfilament reorganization. Binding of the cells to the EGFR-antibody resulted in the formation of a unique cell shape characterized by numerous, actin-based filopodia radiating from the cell body, but without membrane ruffles. There was also a conspicuous circular belt of actin-containing fibers inside the cell margin, and many irregular actin aggregates in the perinuclear area. The morphologies and actin distributions in A-431 cells spread on fibronectin or laminin 2/4 were very different. On fibronectin, cells had polygonal shapes with numerous stress-fibers and thick actin-containing fibers along the cell edges. On laminin-covered substrata, the cells became fusiform and acquired broad leading lamellae with ruffles. In these cells, there were also a few bundles of filaments running the whole length of the cell body, and shorter bundles extending through the leading lamellae towards the membrane ruffles in the cell edge. These effects and those seen with immobilized EGF suggest that different ligand/receptor complexes induce specific reorganizations of the microfilament system.     

Capping of concanavalin a receptors and their association with microfilaments in monolayer grown human fibroblastoid cells

Summary The distribution of ConA binding sites on the surface of normal human fibroblastoid cells grown in monolayer culture was carefully examined. Low concentrations of ConA (between 0.5–5.0 g/ml) were found to induce the ConA receptors to form a single, large cap structure. High concentrations of ConA (between 50–100 g/ml) inhibit cap formation at temperatures above 20° C. Pretreatment of the cells in the cold or with colchicine allows cap formation to occur with high concentrations of ConA. The ConA caps appear to be preferentially localized near the nucleus. Using a double immunofluorescence technique, we have observed actin and myosin molecules concentrated underneath the surface receptor cap in the perinuclear region of the cells. These findings suggest that the binding of ConA to fibroblastoid cells may trigger the transmembrane association of cytoplasmic microfilaments with surface membrane receptors as previously proposed for lymphocytes and other round cells grown in suspension culture.Abbreviations IgG immunoglobulin - Fl fluorescein - Rh rhodamine - PBS 0.1 M phosphate buffer (pH:7.4) plus 0.15 M NaCl - ConA Concanavalin A This work was supported by U.S. Public Health Service Grants AI 15258-01 and CA 22453     

Remodeling of the actin cytoskeleton of target hepatocytes and NK cells during induction of apoptosis.

Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 microM) partly protected against the rearrangement of the actin filaments in the hepatocytes.     

Studies on rabbit lymphocytes in vitro : XVII. Kinetics of reversible concanavalin a stimulation and restimulation of blast transformation after blocking with α-methyl- -mannopyranoside

The stimulation of transformation of rabbit peripheral blood lymphocytes by Concanavalin A (ConA) has a narrow dose optimum, is reversible by α- -methyl-mannopyranoside (MAM) and cultures that have been stimulated and reversed may be restimulated by removal of the blocking saccharide and re-addition of ConA. The kinetics of the stimulatory dose of ConA, the blocking dose of MAM, and the time of stimulation and blocking indicate a competitive binding of lymphocyte receptors and blocking saccharide for ConA. Most of the lymphocytes that respond to ConA become enlarged during the first 16–24 h after stimulation, although fully developed ‘blast’ cell transformation and mitosis do not occur until after approx. 40 h. Lymphocytes that are held in vitro prior to ConA stimulation gradually lose the ability to respond to ConA stimulation (delayed stimulation). Morphologic and metabolic analysis of ConA-stimulated and MAM blocked cultures demonstrate (1) that RNA synthesis gradually decreases in blocked cultures at a time that it is increasing in stimulated cultures; (2) that cells enlarged after ConA stimulation become smaller following MAM inhibition; (3) that the ability of blocked cells to be restimulated by ConA gradually decreases following MAM block (delayed restimulation). Lymphocyte activation requires the continued presence of the stimulant for consumation of the transformation process, and activated cells that have been blocked have a temporary ability to respond to restimulation at a time when cells that have not been preactivated are unable to respond. The requirement of increasing amounts of blocking MAM to reverse stimulation by ConA as the time of contact of ConA with the cells in culture increases is consistent with the concept that internalization, or stripping of mitogen or cellular receptors is the important cellular event initiating transformation. Blocking is achieved by permitting re-externalization of lectin or cell surface receptors. Stimulation requires the continued internalization or stripping of newly formed receptors by reaction with the stimulating mitogen during the entire culture period prior to initiation of DNA synthesis.     

Studies on rabbit lymphocytes in vitro: XVII. Kinetics of reversible concanavalin a stimulation and restimulation of blast transformation after blocking with α-methyl-d-mannopyranoside

The stimulation of transformation of rabbit peripheral blood lymphocytes by Concanavalin A (ConA) has a narrow dose optimum, is reversible by α-d-methyl-mannopyranoside (MAM) and cultures that have been stimulated and reversed may be restimulated by removal of the blocking saccharide and re-addition of ConA. The kinetics of the stimulatory dose of ConA, the blocking dose of MAM, and the time of stimulation and blocking indicate a competitive binding of lymphocyte receptors and blocking saccharide for ConA. Most of the lymphocytes that respond to ConA become enlarged during the first 16–24 h after stimulation, although fully developed ‘blast’ cell transformation and mitosis do not occur until after approx. 40 h. Lymphocytes that are held in vitro prior to ConA stimulation gradually lose the ability to respond to ConA stimulation (delayed stimulation). Morphologic and metabolic analysis of ConA-stimulated and MAM blocked cultures demonstrate (1) that RNA synthesis gradually decreases in blocked cultures at a time that it is increasing in stimulated cultures; (2) that cells enlarged after ConA stimulation become smaller following MAM inhibition; (3) that the ability of blocked cells to be restimulated by ConA gradually decreases following MAM block (delayed restimulation). Lymphocyte activation requires the continued presence of the stimulant for consumation of the transformation process, and activated cells that have been blocked have a temporary ability to respond to restimulation at a time when cells that have not been preactivated are unable to respond. The requirement of increasing amounts of blocking MAM to reverse stimulation by ConA as the time of contact of ConA with the cells in culture increases is consistent with the concept that internalization, or stripping of mitogen or cellular receptors is the important cellular event initiating transformation. Blocking is achieved by permitting re-externalization of lectin or cell surface receptors. Stimulation requires the continued internalization or stripping of newly formed receptors by reaction with the stimulating mitogen during the entire culture period prior to initiation of DNA synthesis.     

Interaction of tumour promoters with epithelial cells in culture : An immunofluorescence study

12-O-tetradecanoyl phorbol-13-acetate (TPA) has a profound and rapid influence on the cytoskeleton of Madin-Darby Canine Kidney (MDCK) cells. Within 10 min, TPA induces a rapid change in morphology, from a flat, cuboidal state to a rounded or elongated morphology in which the cell membranes become convoluted. Concomitant with this morphological change is a rapid dissolution of stress fibres and a redistribution of F-actin from microfilament bundles to a membrane or sub-membranous location. The rearrangement of actin is paralleled by a rearrangement of alpha-actinin and a reduction in the number of vinculin-containing adhesion plaques. Unusual F-actin configurations are often found emanating from a perinuclear location, usually containing alpha-actinin and terminating in a vinculin-containing adhesion plaque. The cytoskeletal rearrangements occur in the presence of inhibitors of protein synthesis or oxidative phosphorylation, but do not occur if glycolysis is also inhibited. The rearrangements are partly abrogated by the presence of cytochalasin B (CB). Despite these dramatic changes in microfilaments the polymerization state of actin remained unaltered after TPA treatment. Furthermore, although changes in the movement of membrane lipids have been reported, no obvious differences in the ability of glycoproteins to redistribute in the plane of the membrane were found as judged by FITC-concanavalin A (conA) induced patching. The rapidity of the morphological response of MDCK cells to TPA indicates that the cytoskeleton is one of the primary targets of TPA, but that tumour promoters differ from RNA tumour viruses in their effect on the state of actin polymerization.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.