Thy-1: more than a mouse pan-T cell marker

Thy-1 (CD90) is a small GPI-anchored protein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells. T cell proliferation and cytokine synthesis in response to Thy-1 cross-linking by specific mAb suggests a role for Thy-1 in mouse T lymphocyte activation. However, a physiological ligand or counterreceptor for murine Thy-1 in the lymphoid compartment has not yet been identified. Thy-1 cross-linking, in the context of strong costimulatory signaling through CD28, results in an activating signal that can at least partially substitute for TCR signaling during mouse T cell activation. Remarkably, Thy-1 cross-linking also results in the potent costimulation of T cells activated through the TCR. This novel dual signaling capacity suggests a possible role for Thy-1 in the maintenance of T cell homeostasis in the absence of TCR triggering, as well as potentiating Ag-induced T cell responses.     

Low density lipoprotein receptor and cation-independent mannose 6- phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells

Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.     

Functionally different GPI proteins are organized in different domains on the neuronal surface

We have investigated the organization, on the plasma membrane and in detergent-insoluble membrane vesicles, of two neuronal glycosylphosphatidylinositol-anchored (GPI) proteins: Thy-1, a negative regulator of transmembrane signalling; and prion protein, whose rapid endocytosis and Cu(2+) binding suggest that it functions in metal ion uptake. Prion protein occurred on the neuronal surface at high density in domains, located primarily at the cell body, which were relatively soluble in detergent. Thy-1, although much more abundantly expressed on neurons, occurred at lower density over much of the surface of neurites (and in lower abundance at the cell body) in domains that were highly resistant to detergent solubilization. Detergent-insoluble membrane vesicles contained Thy-1 at a density similar to that on the neuronal surface. Vesicles containing each protein could be separated by immunoaffinity isolation; lectin binding showed that they were enriched in different glycoproteins. Our results demonstrate a structural diversity of the domains occupied by functionally different GPI proteins.     

Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro.

It was established previously that committed precursors of T cells, which reside in bone marrow and spleen and lack T cell surface differentiation antigens, can be induced by thymopoietin and certain other agents to differentiate rapidly in vitro into T cells bearing typical surface antigens, including Thy-1 and TL (Komuro-Boyse assay). To relate this differentiative step observed in vitro to physiologic events in vivo, a system was devised to trace the migration of precursor cells to the thymus, and their maturation to T cells. Lethally irradiated mice of a TL- strain received spleen cells from TL+ hybrids i.v., and the TL+ population of the thymus was enumerated 13 to 20 days later. Donor TL+ cells first became detectable at 13 days and increased thereafter. Preliminary tests showed that cells capable of migrating to the thymus have a similar density to the cells that are inducible in the Komuro-Boyse assay, this being lower than that of mature of T cells. The thymus-repopulating properties of the donor spleen population were not affected by: 1) pre-treatment in vitro with thymus extract or thymopoietin, which initiates differentiation of T cells precursors, nor b) pre-treatment with anti Thy-1 serum plus complement, which eliminates differentiated T cells. But pre-treatment a) and b) applied in sequence markedly reduced the capacity of spleen cells to repopulate the thymus. These results can be interpreted as follows: induction of Thy-1-TL- precursor cells (pro-thymocyte) in vitro yields Thy-1+TL+ cells (early thymocytes) which have not yet lost their property of repopulating the thymus; therefore, thymus-repopulation was not depleted by treatment a) alone, which induced Thy-1 +TL+ cells, nor by treatment b) alone, which did not affect thymus-repopulation by Thy-1-TL- cells, although treatments a) plus b) did eliminate the newly induced Thy-1+TL+ cells and thus impaired repopulation of the thymus. We conclude that the cell which responds to thymopoietin in the Komuro-Boyse assay by expressing the T cell surface phenotype is the same cell (pro-thymocyte) that normally migrates in vivo from hemopoietic tissues to the thymus and is there induced by thymopoietin to express the phenotype of an early T cell.     

Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.     

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.     

Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition

The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 degrees C undermines the claim that DRMs consist of lipid nanodomains that exist in an L(o) (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg(2+), K(+)) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 degrees C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 degrees C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 degrees C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.     

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.     

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.     

The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein

Thy-1 glycoprotein is a member of a class of proteins which are anchored to the plasma membrane via a covalently bound glycophospholipid. The biosynthesis and anchoring of Thy-1 were investigated in a family of wild-type and mutant (complementation groups A, B, C, E, and F) T lymphomas. The mutants all synthesize Thy-1 but fail to express it on the cell surface. Analysis of the size of D-[2-3H]mannose-labeled dolichol-linked oligosaccharides showed that the class E mutant is the only cell line which does not synthesize dolichol-P-P-Glc3Man9GlcNAc2. Turnover and possible secretion of Thy-1 by mutant T lymphoma cells were documented in D-[2-3H]mannose pulse-chase experiments. The turnover of [3H]Thy-1 for all wild-type cells is considerably slower than for the mutant cells. Class B and E cells release appreciably more [3H]Thy-1 than wild-type cells. Additional experiments were performed to determine the electrophoretic mobility and hydrophobicity of cell-associated and released forms of Thy-1 labeled overnight with [3H]mannose. All wild-type and class A, C, E, and F mutant cells contain a major Triton X-114 binding species of cell-associated [3H]Thy-1. All extracellular [3H]Thy-1 was almost exclusively hydrophilic. The presence of two Thy-1 anchor components, ethanolamine and palmitate, was investigated. Biosynthetic labeling with [3H]palmitic acid showed that all of the wild-type cells but none of the mutants incorporated this anchor precursor into Thy-1. In [3H]ethanolamine-labeling experiments, incorporation was detected in the Thy-1 of all wild-type cells and in two mutants, S1A-b and T1M1-c. Based on the above studies, the phenotype of Thy-1 negative T lymphoma mutants can be re-evaluated. In classes A and F, dolichol-linked oligosaccharides appear normal and no anchor is detected. In class B, dolichol-linked oligosaccharides appear normal, a partial anchor may be present, and a substantial amount of Thy-1 is released. In class C, dolichol-linked oligosaccharides appear normal and a partial anchor may be present. In class E, truncated dolichol-linked oligosaccharides are formed, no anchor is detected, but a substantial amount of newly synthesized Thy-1 is released. These observations are discussed with reference to the possibility that the lesions which characterize the mutants pertain to the biosynthesis of the glycophospholipid moiety of Thy-1.     

Sulfated glycans directly interact with mouse Thy-1 and negatively regulate Thy-1-mediated adhesion of thymocytes to thymic epithelial cells.

Thy-1 is a major brain cell surface glycoprotein of adult mammal species also expressed in rodent thymus. Despite extensive studies, the function(s) of this molecule has remained so far ill defined. We have recently shown that Thy-1 was involved in the adhesion of mouse thymocytes to thymic epithelium through a specific interaction with a heterophilic ligand(s) expressed on the epithelial cell surface. In the present study, we aimed at evaluating the interaction of sulfated glycans with mouse Thy-1, as well as its consequence on Thy-1-mediated thymic lympho-epithelial cell interaction. It was shown that 125I-labeled Thy-1 directly bound to immobilized heparin. Sulfated glycans such as pentosan sulfate, dextran sulfate, and fucoidan were found to strongly inhibit the binding of Thy-1 to heparin. In contrast, chondroitin sulfate, keratan sulfate, and heparan sulfate were not inhibitory. Sulfated glycans (e.g., pentosan sulfate, assayed at a concentration of 50 micrograms/ml) completely blocked the Thy-1-dependent adhesion of T cells to a mouse thymic epithelial cell monolayer. To explore the mechanism of this inhibition, we compared the ability of T cell to adhere to mouse thymic epithelial cell monolayer or to sulfated glycans. Our results suggest that sulfated glycans bind to a Thy-1 site distinct from that with which this molecule interacts with its heterophilic ligand. Moreover, sulfate glycans could modulate the binding of rat mAb directed at spatially distinct Thy-1 epitopes. The present results identified a potential mechanism regulating Thy-1-mediated lympho-epithelial cell adhesion.     

Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1.

To examine the extent to which protein structure and tissue-type influence glycosylation, we have determined the oligosaccharide structures at each of the three glycosylation sites (Asn-23, 74 and 98) of the cell surface glycoprotein Thy-1 isolated from rat brain and thymus. The results show that there is tissue-specificity of glycosylation and that superimposed on this is a significant degree of site-specificity. On the basis of the site distribution of oligosaccharides, we find that no Thy-1 molecules are in common between the two tissues despite the amino acid sequences being identical. We suggest, therefore, that by controlling N-glycosylation a tissue creates an unique set of glycoforms (same polypeptide but with oligosaccharides that differ either in sequence or disposition). The structures at each of the three sites were also determined for the thymocyte Thy-1 that binds to lentil lectin (Thy-1 L+) and for that which does not (Thy-1 L-). Segregation of intact thymus Thy-1 into two distinct sets of glycoforms by lentil lectin was found to be due to the structures at site 74. Analysis of oligosaccharide structures at the 'passenger' sites (23 and 98) suggests that either Thy-1 L+ and Thy-1 L- molecules are made in different cell-types or that the biosynthesis of oligosaccharides at one site is influenced by the glycosylation at other sites.     

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.     

Thy-1 variants of mouse lymphomas: biochemical characterization of the genetic defect

The surface proteins of Thy-1 positive mouse lymphomas and Thy-1 negative variants were labeled by lactoperoxidase-catalyzed iodination and characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by immunological studies. The loss of the serologically defined Thy-1 antigenic determinant correlated with the absence of a radioactive band corresponding to a T lymphocyte-specific surface glycoprotein, T25, on autoradiographs of the iodinated proteins of Thy-1 negative variants. Reexpression of Thy-1 on hybrid cells derived from fusions of complementary Thy-1 negative variants correlated with the reappearance of T25. Quantitative absorption studies using an antiserum which specifically recognized T25 confirmed that the Thy-1 negative variants have no detectable T25 (<0.03 of that of the Thy-1 positive lines) on their surface.Biosynthetic labeling studies revealed that none of the Thy-1 negative variants synthesized T25. However, synthesis of an immunologically cross-reactive molecule could be detected in two variants. On the basis of these results, we propose a model which describes a possible structure of the Thy-1 antigenic determinant and explains the biochemical nature of the genetic lesions leading to the loss of Thy-1 in these variants.     

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.     

Connexin43 associated with an N-cadherin-containing multiprotein complex is required for gap junction formation in NIH3T3 cells

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membrane-membrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of connexin43 (Cx43alpha1) gap junction protein with N-cadherin, p120, and other N-cadherin-associated proteins at regions of cell-cell contact. We also found that Cx43alpha1, N-cadherin, and N-cadherin-associated proteins were coimmunoprecipitated by antibodies to either Cx43alpha1, N-cadherin, or various N-cadherin-associated proteins. These findings suggest that Cx43alpha1 and N-cadherin are coassembled in a multiprotein complex containing various N-cadherin-associated proteins. Studies using siRNA knockdown indicated that cell surface expression of Cx43alpha1 required N-cadherin, and conversely, N-cadherin cell surface expression required Cx43alpha1. Pulse-chase labeling and cell surface biotinylation experiments indicated that in the absence of N-cadherin, Cx43alpha1 cell surface trafficking is blocked. Surprisingly, siRNA knockdown of p120, an N-cadherin-associated protein known to modulate cell surface turnover of N-cadherin, reduced N-cadherin cell surface expression without altering Cx43alpha1 expression. These observations suggest that in contrast to the coregulated cell surface trafficking of Cx43alpha1 and N-cadherin, N-cadherin turnover at the cell surface may be regulated independently of Cx43alpha1. Functional studies showed gap junctional communication is reduced and cell motility inhibited with N-cadherin or Cx43alpha1 knockdown, consistent with the observed loss of both gap junction and cadherin contacts with either knockdown. Overall, these studies indicate that the intracellular coassembly of connexin and cadherin is required for gap junction and adherens junction formation, a process that likely underlies the intimate association between gap junction and adherens junction formation.     

Monoclonal antibody against actin cross-reacts with the Thy-1 molecule and inhibits lymphocyte function-associated antigen-1 dependent cell-cell interaction of T cells.

To assess the molecular mechanisms involved in cell-cell interactions of T cells, we produced a mAb that could block PMA-induced homotypic T cell aggregation. The established mAb (TN14 mAb) showed an inhibitory effect on T cell aggregation as large as that of mAb against lymphocyte function-associated Ag-1. TN14 mAb showed a strong reactivity with cell surface molecules on T cells, whereas it did not react with any cell surface Ag on macrophages, B cells, bone marrow cells, or WEHI 164 cells, which were used for immunization. However, all cell lysates could react with TN14 mAb in Western blotting analysis, indicating that TN14 mAb recognized some cytoplasmic protein. From biochemical analysis, TN14 mAb was demonstrated to react with the 43-kDa cytoskeletal protein actin. Moreover, from an immunoprecipitation study, TN14 mAb was demonstrated to cross-react with the Thy-1 molecule on T cells. The binding activity of TN14 mAb with the Thy-1 molecule on T cells was strongly blocked by the addition of purified actin, indicating that TN14 mAb recognized an actin-related epitope of the Thy-1 molecule. TN14 mAb blocked not only T cell aggregation but also cell-mediated cytotoxicity by CTL. These results strongly support the hypothesis that the Thy-1 molecule, a member of the Ig superfamily, may play an important role in cell-cell interactions of T cells.     

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6- phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti- phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.     

Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.