Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.     

Cytochalasin D disrupts the restricted localization of N-CAM,but not of L1, at sites of Schwann cell—neurite and Schwann cell–Schwann cell contact in culture

The neural recognition molecules L1 and N-CAM have been shown to be preferentially localized at sites of Schwann cell-to-neurite and Schwann cell-to-Schwann cell contact in vitro. In the present study, we investigated the mechanisms underlying the restricted expression of these molecules at the Schwann cell surface, focusing on the possible role of actin filaments. Co-cultures consisting of Schwann cells from newborn mice and explants of dorsal root ganglia from chicken embryos were maintained in the absence or presence of cytochalasin D, an agent disrupting actin filaments. Immunoelectron microscopy with mouse-specific antibodies was carried out to quantify the restricted localization of L1 and N-CAM at the Schwann cell surface in contact with neurites. After 2 days of co-culturing in the absence of cytochalasin D, approximately 65% of the cell–cell contacts showed a restricted immunoreactivity for L1 and N-CAM. The accumulation of L1 at contact sites was unchanged in cytochalasin D-treated co-cultures, while the agent strongly reduced the restricted localization of N-CAM to 20% of all cell–cell contacts. The disruption of N-CAM accumulation appeared to be rapid and occurred within 5 h of cytochalasin D treatment. These results indicate that the restricted localization of N-CAM, but not of L1, is sensitive to cytochalasin D treatment, suggesting a dependence on the integrity of the actin network. Thus, different mechanisms may regulate the subcellular distribution of cell adhesion molecules in Schwann cells.     

THE THEORETICAL RESPONSE OF LIVING CELLS TO CONTACT WITH SOLID BODIES

The theoretical behavior of a hypothetical fluid cell in contact with flat and curved solid surfaces is discussed from the point of view of surface tension. An equation is derived for calculating the equilibrium position of the cell on a flat surface in terms of the surface tensions between the cell and the plasma, the plasma and the solid surface, and the solid surface and the cell. It is shown that the same equilibrium is predicted from consideration of the contact angle between the cell and the solid body. The relative surface energy has been calculated at various stages in the ingestion of a solid particle by a fluid cell four times as large in diameter, and it is thus shown that no particle will be ingested until the surface tensions are such that the cell would spread to infinity on a flat surface of the same substance. Here again the same equilibrium is predicted from considerations of the contact angle. The adhesiveness of blood cells to solid substances is shown to be a pure surface tension phenomenon, but in most reactions between living cells and solid bodies the fluidity of the protoplasm is also a factor of prime importance. The frequent occurrence of adhesiveness as a property of cells in contact with solid bodies is due in part to the fact that, by so adhering, the surface area of the cell not touching the solid is decreased.     

Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.     

Effects of substratum morphology on cell physiology

Among the host of substratum properties that affect animal cell behavior, surface morphology has received relatively little attention. The earliest effect of surface morphology on animal cells was discovered almost a century ago when it was found that cells became oriented in response to the underlying topography. This phenomenon is now commonly known as contact guidance. From then until very recentrly, little progress has been made in understanding the role of surface morphology on cell behavior, primarily due to a lack of defined surfaces with uniform morphologies. This problem has been solved recently with the development of photolithographic techniques to prepare substrata with well defined and uniform surface morphologies. Availability of such surfaces has facilitated systematic in vitro experiments to study influence of surface morphology on diverse cell physiological aspects such as adhesion, growth, and function. For example, these studies have shown that surfaces with uniform multipls parallel grooves can enhance cell adhesion by confining cells in grooves and by mechanically interlocking them. Several independent studies have demosterated that cell shape is a major determinant of cell growth and function. Because surface morphology has been shown to modulate the extent of cell spreading and cell shape, its effects on cell growth and function appear to be mediated via this biological coupling between cell shape and function. New evidence in the cell biology literature is emerging to suggest that surface morphology could affect other cell behavioral properties such as post-translational modifications. Further elucidation of such effects will enable better designs for implant and cell culture substrata.     

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.     

Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.     

CELL CONTACT DEPENDENCE OF SURFACE GALACTOSYLTRANSFERASE ACTIVITY AS A FUNCTION OF THE CELL CYCLE

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle. Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type. The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.     

Extracellular nicotinamide adenine dinucleotide induces t cell apoptosis in vivo and in vitro.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3(+), CD4(+), and CD8(+) cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69(+) T cells.     

Transcytosis in MDCK cells: identification of glycoproteins transported bidirectionally between both plasma membrane domains

MDCK cells display fluid-phase transcytosis in both directions across the cell. Transcytosis of cell surface molecules was estimated by electron microscopic analysis of streptavidin-gold-labeled frozen sections of biotinylated cells. Within 3 h, approximately 10% of the surface molecules, biotinylated on the starting membrane domain, were detected on the opposite surface domain irrespective of the direction of transcytosis. This suggests that the transcytosis rates for surface molecules are equal in both directions across the cell as shown previously for fluid-phase markers. A biochemical assay was established to identify transcytosing glycoproteins in MDCKII-RCAr cells, a ricin- resistant mutant of MDCK. Due to a galactosylation defect, surface glycoproteins of these cells can be labeled efficiently with [3H]galactose. Transcytosis of [3H]galactose-labeled glycoproteins to the opposite membrane domain was detected by surface biotinylation. Detergent-solubilized glycoproteins derivatized with biotin were adsorbed onto streptavidin-agarose and separated by SDS-PAGE. A subset of the cell surface glycoproteins was shown to undergo transcytosis. Transport of these glycoproteins across the cell was time and temperature dependent. By comparative two-dimensional gel analysis, three classes of glycoproteins were defined. Two groups of glycoproteins were found to be transported unidirectionally by transcytosis, one from the apical to the basolateral surface and another from the basolateral to the apical surface. A third group of glycoproteins which has not been described previously, was found to be transported bidirectionally across the cell.     

B cell-associated LFA-1 and T cell-associated ICAM-1 transiently cluster in the area of contact between interacting cells.

TNP-specific B cells interact with carrier-specific T hybridoma cells in an antigen-specific, MHC-restricted manner. The formation of T cell/B cell conjugates is time and temperature dependent and results in the formation of a broad area of close contact between the interacting cells. In order to determine which surface molecules on the two cells cluster at the interaction site. T cell/B cell conjugates were formalin-fixed at different times following conjugation and were stained with antibodies directed against cell surface molecules. Results of these studies indicate that the alpha- and beta-subunits of LFA-1 on B cells transiently cluster in the area of cell contact. Maximum clustering of LFA-1 occurs at 45 min, after which time LFA-1 redistributes on the surface of the B cells. Several other B cell-associated molecules (MHC Class II, ICAM-1, Ig, B220, J11D, or CD23) do not cluster at the interaction site at any time point. T cell-associated LFA-1 does not cluster with any specific pattern, but ICAM-1 does. Maximum clustering of ICAM-1 occurs 60 to 90 min after intercellular contact. After this time, ICAM-1 redistributes on the surface of the T cells. Although both the alpha- and beta-subunits of LFA-1 cluster at the interaction site on B cells, antibodies recognizing these subunits differ in their ability to affect conjugation. One antibody recognizing the alpha chain of LFA-1 (M17/4.2) inhibits T-cell/B cell conjugation, whereas another antibody that also recognizes the alpha chain-(G-48) enhances conjugation. In contrast, an antibody that recognizes LFA-1 beta (M18/2.a.8) has no effect. An antibody that recognizes ICAM-1 (YN/1.7), the ligand for LFA-1, inhibits conjugation. These data show that, during T cell/B cell interaction. LFA-1 on B cells and ICAM-1 on T cells transiently cluster with similar, albeit not identical, kinetics to the site of cell-cell contact.     

Dynamic aspects of adhesion receptor function — integrins both twist and shout

The recognition of extracellular molecules by cell surface receptors is the principal mechanism used by cells to sense their environment. Consequently, signals transduced as a result of these interactions make a major contribution to the regulation of cellular phenotype. Historically, particular emphasis has been placed on elucidating the intracellular consequences of growth factor and cytokine binding to cells. In addition to these interactions, however, cells are usually in intimate contact with a further source of complex structural and functional information, namely immobilised extracellular matrix and/or cell surface adhesion proteins. A key question in recent years has been whether cells use the myriad of adhesion protein-receptor interactions purely for structural and migratory function, or whether these interactions also make a more varied contribution to cell phenotype. Here we review dynamic aspects of the function of one major class of adhesion receptor, the integrins. In particular, we focus on the evidence for shape changes in integrin molecules, the mechanisms responsible for regulating ligand binding, and the signals transduced following integrin occupancy.     

Muscle cell outside and inside: the nascent approach of a clinician.

Understanding muscle cell in disease and health is an unfinished process. Following the lead of Jaime Alvarez, I have had the opportunity of working on two complementary approaches to this field. One is the study of muscle cell surface molecules. Both synaptic muscle molecules, such as the asymmetric form of acetylcholinesterase, and extrasynaptic molecules, such as the extracellular matrix proteoglycans, are regulated by the motor nerve activity. This illustrates one of Jaime's teachings: cell phenotypes are a dynamic process that reflects the influence of other cells (Alvarez, 2001). Proteoglycans have many functions, including growth factor receptors. Studying them in muscular dystrophy will contribute to the comprehension of the muscle regeneration failure, characteristic of this disease. Muscle cells are highly dependent upon energy production, and the mitochondriae produce most of it. These organelles are unique in having their own genome. Mutations in these genes have recently been recognized as the cause of human disease and originally in muscle pathology. The physiopathology of these diseases is summarized here.     

Dynamics of adhesion molecule domains on neutrophil membranes: surfing the dynamic cell topography

Lateral organization and mobility of adhesion molecules play a significant role in determining the avidity with which cells can bind to target cells or surfaces. Recently, we have shown that the lateral mobility of the principal adhesion molecules on neutrophils is lower for rolling associated adhesion molecules (RAAMs: L-selectin and PSGL-1) than for β₂ integrins (LFA-1 and Mac-1). Here we report that all four adhesion molecules exhibit distinct punctate distributions that are mobile on the cell surface. Using uniform illumination image correlation microscopy, we measure the lateral mobility of these topologically distinct domains. For all four molecules, we find that diffusion coefficients calculated from domain mobility agree with measurements we made previously using fluorescence recovery after photobleaching. This agreement indicates that the transport of receptors on the surface of the resting neutrophil is dominated by the lateral movement of domains rather than individual molecules. The diffusion of pre-assembled integrin domains to zones of neutrophil/endothelial contact may provide a mechanism to facilitate high avidity adhesion during the earliest stages of firm arrest.     

Cell surface and cytokine phenotypes of skin immunocompetent cells involved in ultraviolet-induced immunosuppression

Immunocellular migrations out of and into the skin and modulations of functional cell surface molecules on antigen-presenting cells (APCs), as well as their immunoregulatory cytokine production, are important factors involved in the mechanism of UV-induced immunosuppression and tolerance. Of particular interest here are the effects of low-dose UVB exposures that can suppress the ability of a contact sensitizer to induce contact hypersensitivity (CHS) through the site (local immunosuppression) without inducing suppression of CHS induced through skin distant to the UV exposure. Such UV-irradiated skin has many changes with respect to composition of immunocompetent cells and cytokine production. After UV exposure, Langerhans cells/dendritic cells migrate from the skin to draining lymph nodes (DLNs) as they do from contact sensitizer-applied normal skin. On the other hand, UV causes monocytic/macrophagic cells to infiltrate into the dermis and then into the epidermis; these can also be shown to be induced by contact sesitizers to migrate to DLNs. Alterations in cell surface and immunoregulatory cytokine phenotypes of the cutaneous APCs in both the skin and DLNs are critical for CHS suppression and tolerance induction. Here we describe the phenotypic changes of immunocompetent cells in UV-irradiated skin in regard to CHS suppression and tolerance and methodologies to approach this area.     

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.     

Marginal product pricing in the ecosystem.

Surface membrane biosynthesis and turnover is reviewed focusing mainly on the fate of cell surface constituents after they terminated their sojourn as part of a functional cell structure. The different experimental approaches to study this problem are described and original data are presented on the turnover of surface membrane constituents of chicken embryo cells in culture. It is proposed that as a consequence of surface membrane turnover, certain surface macromolecules are continuously shed from cells. The size and charge of these molecules was found to be identical to molecules released from cells by mild trypsin treatment. The term shedding is proposed for this process which is assumed to occur both in vitro and in vivo. Many systems in which shedding of cell surface constituents is clearly demonstrated or can be tentatively suggested are described. The biological significance of cell surface carbohydrate containing macromolecules and the possible role of these shed cellular entities is discussed.