Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P(2)-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P(2)-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P(2), Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P(2) in a Ca(2+)/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P(2)-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.     

The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway

Sorting signals for apically destined proteins are highly diverse and can be present within the luminal, membrane-associated, and cytoplasmic domains of these proteins. A subset of apical proteins partition into detergent-resistant membranes, and the association of these proteins with glycolipid-enriched microdomains or lipid rafts may be important for their proper targeting. Recently, we observed that raft-associated and raft-independent apical proteins take different routes to the apical surface of polarized Madin-Darby canine kidney cells (Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007) EMBO J. 26, 3737-3748). Here we reconstituted in vitro the export of raft-associated and raft-independent markers staged intracellularly at 19 degrees C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19 degrees C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.     

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.     

Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins

Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft-associated proteins are detected. In this report, we demonstrate that by incorporating 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) crosslinking and freezing at -80°C into the membrane raft isolation procedure of HaCaT cells, both membrane raft-associated proteins caveolin-1 and Fas receptor are able to be reproducibly isolated into a single fraction containing the membrane rafts of the cells.     

The SPFH domain-containing proteins: more than lipid raft markers

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.     

The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.     

Lipid rafts: cell surface platforms for T cell signaling

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.     

Lipid rafts: structure, function and role in HIV, Alzheimer's and prion diseases

The fluid mosaic model of the plasma membrane has evolved considerably since its original formulation 30 years ago. Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids (GPLs) and cholesterol, but a mosaic of domains with unique biochemical compositions. Among these domains, those containing sphingolipids and cholesterol, referred to as membrane or lipid rafts, have received much attention in the past few years. Lipid rafts have unique physicochemical properties that direct their organisation into liquid-ordered phases floating in a liquid-crystalline ocean of GPLs. These domains are resistant to detergent solubilisation at 4 degrees C and are destabilised by cholesterol- and sphingolipid-depleting agents. Lipid rafts have been morphologically characterised as small membrane patches that are tens of nanometres in diameter. Cellular and/or exogenous proteins that interact with lipid rafts can use them as transport shuttles on the cell surface. Thus, rafts act as molecular sorting machines capable of co-ordinating the spatiotemporal organisation of signal transduction pathways within selected areas ('signalosomes') of the plasma membrane. In addition, rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the [beta]-amyloid peptide associated with Alzheimer's disease. The possibility of modulating raft homeostasis, using statins and synthetic sphingolipid analogues, offers new approaches for therapeutic interventions in raft-associated diseases.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.     

Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.     

The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Regulation by distinct cysteine-rich domains

SNAP-25 and its ubiquitously expressed homologue, SNAP-23, are SNARE proteins that are essential for regulated exocytosis in diverse cell types. Recent work has shown that SNAP-25 and SNAP-23 are partly localized in sphingolipid/cholesterol-rich lipid raft domains of the plasma membrane and that the integrity of these domains is important for exocytosis. Here, we show that raft localization is mediated by a 36-amino-acid region of SNAP-25 that is also the minimal sequence required for membrane targeting; this domain contains 4 closely spaced cysteine residues that are sites for palmitoylation. Analysis of endogenous levels of SNAP-25 and SNAP-23 present in lipid rafts in PC12 cells revealed that SNAP-23 (54% raft-associated) was almost 3-fold more enriched in rafts when compared with SNAP-25 (20% raft-associated). We report that the increased raft association of SNAP-23 occurs due to the substitution of a highly conserved phenylalanine residue present in SNAP-25 with a cysteine residue. Intriguingly, although the extra cysteine in SNAP-23 enhances its raft association, the phenylalanine at the same position in SNAP-25 acts to repress the raft association of this protein. These different raft-targeting signals within SNAP-25 and SNAP-23 are likely important for fine-tuning the exocytic pathways in which these proteins operate.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.     

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.