Glycolipid-enriched Membrane Microdomains Research Articles

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells

Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.

TNF differentially regulates ganglioside biosynthesis and expression in breast cancer cell lines.

Gangliosides are glycosphingolipids concentrated in glycolipid-enriched membrane microdomains. Mainly restricted to the nervous system in healthy adult, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. GD3 synthase (GD3S), the key enzyme that controls the biosynthesis of complex gangliosides, was shown to be over-expressed in Estrogen Receptor (ER)-negative breast cancer tumors, and associated with a decreased overall survival of patients. We previously demonstrated that GD3S expression in ER-negative breast cancer cells induced a proliferative phenotype and an increased tumor growth. In addition, our results clearly indicate that Tumor Necrosis Factor (TNF) induced GD3S over-expression in breast cancer cells via NFκB pathway. In this study, we analyzed the effect of TNF on ganglioside biosynthesis and expression in breast cancer cells from different molecular subtypes. We showed that TNF up-regulated the expression of GD3S in MCF-7 and Hs578T cells, whereas no change was observed for MDA-MB-231. We also showed that TNF induced an increased expression of complex gangliosides at the cell surface of a small proportion of MCF-7 cells. These results demonstrate that TNF differentially regulates gangliosides expression in breast cancer cell lines and establish a possible link between inflammation at the tumor site environment, expression of complex gangliosides and tumor development.

Palmitoylated claudin7 captured in glycolipid-enriched membrane microdomains promotes metastasis via associated transmembrane and cytosolic molecules.

In epithelial cells claudin7 (cld7) is a major component of tight junctions, but is also recovered from glycolipid-enriched membrane microdomains (GEM). In tumor cells, too, cld7 exists in two stages. Only GEM-located cld7, which is palmitoylated, promotes metastasis. Searching for the underlying mechanism(s) revealed the following.The metastatic capacity of the rat pancreatic adenocarcinoma cell line ASML is lost by a knockdown (kd) of cld7 and is not regained by rescuing cld7 with a mutated palmitoylation site (cld7mPalm). ASML-cld7kd and ASML-cld7mPalm cells show reduced motility and invasiveness. This is due to cld7, but not cld7mPalm associating with α6β4, ezrin, uPAR and MMP14, which jointly support motility and invasion. Palmitoylated cld7 also is engaged in drug resistance by repressing Pten, allowing activation of the antiapoptotic PI3K/Akt pathway. An association of cld7mPalm with the major Pten phosphorylating kinases does not restore apoptosis resistance as phosphorylated Pten is not guided towards GEM to compete with non-phosphorylated Pten. The pathway whereby palmitoylated cld7 supports expression of several EMT genes and nuclear translocation of EMT transcription factors remains to be unraveled. An association with Notch, reduced in ASML-cld7mPalm cells, might be the starting point. Finally, GEM-located, palmitoylated cld7 associates with several components of vesicle transport machineries engaged in exosome biogenesis.Taken together, prerequisites for cld7 acting as a cancer-initiating cell marker are GEM location and palmitoylation, which support a multitude of associations and integration into exosomes. The latter suggests palmitoylated cld7 contributing to message transfer via exosomes.

Bioenergetics of stomatin-like protein-2-deficient T cells reveal a regulatory role for this protein in immunometabolism (P1173)

Abstract Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein identified in proteome analysis of glycolipid-enriched membrane microdomains of mammalian cells, and modulates T cell activation. To study SLP-2 function we generated T cell-specific SLP-2 knockout (SLP-2 T-K/O) mice since conventional SLP-2 knockout mice were embryonic lethal at the pre-implantation stage. These mice showed reduced CD4+ T cell responses and decreased respiratory complex I and II+III activities. Our mechanistic studies suggest SLP-2 recruits prohibitins to cardiolipin (CL)-enriched domains in the mitochondrial inner membrane to form specialized CL-enriched membrane microdomains, required for optimal cellular respiration. We hypothesized SLP-2 functions in this manner to regulate the bioenergetics of T cells. Seahorse XF analysis revealed that although SLP-2 T-K/O cells showed unaltered total cellular and mitochondrial oxygen consumption rates (OCRs), they had significantly greater non-mitochondrial OCR, mitochondrial OCR uncoupled from ATP synthesis, and greater reliance on glycolysis (71% of mice). Surprisingly, SLP-2 T-K/O cells exhibited greater spare respiratory capacity, a phenomenon not due to differential sensitivities of cells to uncoupling agents. This new immunometabolic phenotype suggests SLP-2 may regulate assembly of functional respiratory supercomplexes. Bioenergetic and biochemical analyses of mitochondria are underway to establish the regulatory role of SLP-2 in T cell metabolism.

In vivo induced deletion of the mitochondrial protein stomatin-like protein-2 is associated with development of an inflammatory motor deficit disorder (54.10)

Abstract Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein identified in proteome analysis of glycolipid-enriched membrane microdomains of mammalian cells, including lymphocytes. Our preliminary data suggest SLP-2 recruits prohibitins to cardiolipin-enriched domains in the mitochondrial inner membrane to form specialized membrane microdomains, required for optimal cellular respiration. We hypothesized that deletion of the SLP-2 gene will cause defective mitochondrial function that may result in organ-selective disease. We found that a conventional SLP-2-deficient mouse was embryonic lethal at the pre-implantation stage. Thus we generated viable tamoxifen-inducible systemic SLP-2 knockout mice. Administration of tamoxifen (3mg/40g; 10 days; i.p.) resulted in a motor phenotype characterized by poorer SHIRPA scores, weight loss and death (68% at day 28, p<0.05). This phenotype correlated with SLP-2 gene deletion and decreased protein levels in select tissues. Histological analysis revealed multifocal myofibrillar degeneration of the diaphragm with a mononuclear inflammatory infiltrate. In addition, apoptosis throughout the exocrine pancreas and pancreatic atrophy due to loss of normal acinar tissue were observed. In conclusion, in vivo SLP-2 deletion induces a mitochondrial disease-like phenotype, suggesting this protein is required for optimal metabolic function. SLP-2-deficient mice may be useful to assess the function of SLP-2 in vivo and to study mitochondrial diseases.

Transfer of the glycosylphosphatidylinositol-anchored 5'-nucleotidase CD73 from adiposomes into rat adipocytes stimulates lipid synthesis.

In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H(2)O(2), palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMP-degrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time- adiposome concentration- and signal (H(2)O(2) > glimepiride > palmitate)-dependent fashion. The salt- and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H(2)O(2), glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue.

Inhibition of lipolysis by adiposomes containing glycosylphosphatidylinositol-anchored Gce1 protein in rat adipocytes

Small membrane vesicles released from large adipocytes and harbouring the glycosylphosphatidylinositol-anchored (GPI-) AMP-degrading protein CD73 have previously been demonstrated to stimulate the signal-induced esterification of free fatty acids into neutral lipids suggesting a role of these so-called adiposomes (ADIP) in the paracrine regulation of lipid metabolism in the adipose tissue. Here the involvement of another constituent GPI-protein of ADIP, the cAMP-degrading protein Gce1 in the signal-induced inhibition of lipolysis was investigated in primary rat adipocytes. Incubation of small, and to a lower degree, large adipocytes with ADIP inhibited lipolysis and increased its sensitivity toward inhibition by H2O2, the anti-diabetic drug glimepiride and palmitate. This was accompanied by the transfer of Gce1 from the ADIP to detergent-insoluble glycolipid-enriched plasma membrane microdomains (DIGs) and its subsequent translocation to cytoplasmic lipid droplets (LD) of the acceptor adipocytes. The translocation from DIGs to LD rather than the transfer from ADIP to DIGs of Gce1 was stimulated by H2O2 > glimepiride > palmitate. Both transfer and translocation led to salt- and carbonate-resistant association of Gce1 with DIGs and LD, respectively, and relied on the structural integrity of the DIGs and GPI anchor of Gce1. In conclusion, the trafficking of GPI-proteins from ADIP of donor adipocytes via DIGs to LD of acceptor adipocytes mediates paracrine regulation of lipolysis within adipose tissue.

The cell–cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.

Mite-antigen Stimulates MAL Expression in Peripheral Blood T Cells of Mite-sensitive Subjects

Differential gene expression in CD3 + T cells from allergic patients stimulated with allergen will provide important information on the responses of T cells. After stimulation of peripheral blood mononuclear cells (PBMCs) with mite extracts, levels of gene transcription were examined in CD3 + T cells from allergic patients. Stimulation of PBMCs from mite specific IgE positive subjects resulted in specific upregulation of MAL transcription levels that was mediated by IL-4 secretion. The MAL protein in IL-4 stimulated primary T cells preferentially localized in glycolipid-enriched membrane (GEM) microdomains. When MAL was exogenously expressed in primary T cells, CD3ζ was concomitantly enriched, along with the expression of MAL, in GEM microdomains. GEMs are important for the formation and stabilization of TCR signaling complexes. Therefore, MAL may play a role in the formation of GEMs in activated T cells and the high expression of MAL may contribute to Th2 immune response.

CD44 variant isoforms associate with tetraspanins and EpCAM

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-β-cyclodextrin (MβCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell–matrix and cell–cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7–EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.

Lipid rafts, major histocompatibility complex molecules, and immune regulation

Glycolipid-enriched membrane microdomains (“rafts”) are critical sites for signal transduction and other processes such as intracellular transport. While the participation of T-cell rafts in the formation of the immunological synapse is well established, the role of rafts on antigen-presenting cells (APCs) as well as the relationship between these domains and major histocompatibility complex (MHC) molecules is less clearly defined. We therefore investigated whether MHC class I or II molecules are found in rafts of the human macrophage-monocytic cell line U937. We detected the preferential localization of MHC class II, but not class I, molecules in rafts. Furthermore, raft disruption resulted in a decrease in constitutive protein tyrosine phosphorylation events in U937 cells. Our findings are reviewed in the context of results from other groups who also found important associations of MHC class II molecules with APC rafts. Additional, and at times contradictory, findings by others regarding the relationship between rafts and MHC molecules are also discussed. It is concluded that class II MHC molecules can localize in rafts of APCs and that this localization may be relevant for APC function and thus immune regulation.

Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells.

This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP-76, Gads, Vav and ZAP70. We show that both SLP-76 and ZAP70 co-immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co-immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP-76, Gads and Vav associate independently of each other to different domains or regions, of Shb. Overexpression of an SH2 domain-defective Shb causes diminished phosphorylation of SLP-76 and Vav and consequently decreased activation of c-Jun kinase upon T cell receptor (TCR) stimulation. Shb was also found to localize to glycolipid-enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation. Our results indicate that upon TCR stimulation, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP-76-Gads-Vav complex to the T cell receptor zeta-chain and ZAP70.

Anchorage of HIV on Permissive Cells Leads to Coaggregation of Viral Particles with Surface Nucleolin at Membrane Raft Microdomains

The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process.

The Src family kinase Fyn mediates signals induced by TCR antagonists.

FcR nonbinding anti-CD3 epsilon mAbs elicit partial TCR signaling that leads to T cell unresponsiveness and tolerance in vivo. In this study, the membrane-proximal events that promote T cell inactivation by FcR nonbinding anti-CD3 mAbs were examined. In the context of FcR nonbinding anti-CD3, TCR complexes did not aggregate and failed to translocate into glycolipid-enriched membrane microdomains. Furthermore, FcR nonbinding anti-CD3 mAbs induced tyrosine phosphorylation of the Fyn substrate Cbl, but not the ZAP-70 substrate linker for activation of T cells. Overexpression of Fyn, but not Lck, restored the mitogenicity of FcR nonbinding anti-CD3 in primary T cells. Taken together, these results suggest that Fyn mediates the partial signaling induced by TCR antagonists.

C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal.

The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.

Signalling via MHC class II molecules modifies the composition of GEMs in APC.

Major histocompatibility complex (MHC) class II molecules are responsible for peptide presentation to helper T lymphocytes and as such play an essential role in the immune response. These molecules transmit intracellular signals leading to diverse consequences in B lymphocytes including proliferation and apoptosis. Recent studies have revealed that glycolipid enriched membrane microdomains (GEMs) behave as signalling platforms for a variety of lymphocyte receptors. We have quantified human leucocyte antigen (HLA)-DR molecules localized in GEMs in human B lymphocytes. Use of a model imitating the interaction of HLA-DR with a T-cell receptor (TCR) modified the constituents of the HLA-DR-enriched GEMs. Confocal microscopy demonstrated a recruitment of HLA-DR and the ganglioside GM1 at the site of HLA-DR interaction with the stimulating ligand. Moreover, cholesterol depletion efficiently impaired this recruitment. Co-localizing proteins detected in HLA-DR-enriched GEMs include protein kinase C (PKC)-delta and actin. These data reveal that MHC class II antigens are localized in GEMs in mature human B lymphocytes and indicates that the formation of the immunological synapse regulates the composition of HLA-DR enriched GEMs in the antigen presenting cell (APC).

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56lck (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.

Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells.

T cell activation is accompanied by actin-mediated receptor clustering and reorganization of lipid rafts. It has been suggested that costimulatory molecules might be involved in these processes. We here provide evidence that engagement of the adhesion molecule CD44 initiates cytoskeletal rearrangement and membrane reorganization in T cells. Cross-linking of CD44 on a T helper line was accompanied by adhesion, spreading and actin bundle formation. These processes were energy dependent and required an intact actin and microtubule system. They involved the small GTPase Rac as evidenced by the absence of spreading in cells overexpressing a dominant negative form of Rac. The CD44 initiated reorganization of the cytoskeleton was associated with the recruitment of CD44 and the associated tyrosine phosphokinases p56(lck) and p59(fyn) into glycolipid enriched membrane microdomains (GEM). We interpret the data in the sense that CD44 functions as a costimulatory molecule in T cell activation by inducing actin cytoskeletal rearrangements and membrane protein and lipid reorganization including its association with GEMs. Due to the association of CD44 with lck and fyn this colocalization with the TCR allows an abundant provision of these kinases, which are essential to initiate the TCR signaling cascade.

T-lymphocyte coactivator molecules.

T-cell recognition and activation occurs within a specialized area of contact known as the immunologic synapse, localized to areas of glycolipid-enriched membrane microdomains. Within this area, T-cell activation is dependent not only upon specific recognition of peptide antigen embedded within molecules of the major histocompatibility complex, but also on a variety of costimulatory receptors and interactions. Engagement of T-cell receptor (TCR) with antigen alone will induce T-cell unresponsiveness; ligation of the coreceptor CD28 will prevent the induction of unresponsiveness. Novel costimulatory molecules belonging to both the CD28 and TNF/TNFR superfamilies have recently been identified. These receptors appear to act at different stages of T-cell differentiation and activation, have been shown to play a role in promoting different T-cell effector functions, and are important for B-cell differentiation and function.

Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling.

Two hematopoietic-specific adapters, src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224-244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224-244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.