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The role of lipid rafts in signalling and membrane trafficking in T lymphocytes

Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049-Madrid, Spain

View larger version (32K): [in a new window]   Fig. 1. Model of lipid-raft structure and function in biological membranes. (A) Rafts are membrane microdomains formed by high concentrations of sphingolipids (dark-brown-headed structures) and cholesterol (red bean-shaped structures) immersed in a phospholipid-rich (light-brown-headed structures) environment. Glycolipids and sphingomyelin are restricted to the outer leaflet of the bilayer, whereas cholesterol and phospholipids are in both leaflets. Note that lipids in the rafts usually have long and saturated fatty acyl chains (red two-legged shapes), whereas those in lipids excluded from these microdomains are shorter and unsaturated (green two-legged shapes). (B) Principles of selective recruitment of proteins in rafts. Recruitment of membrane proteins in phospholipid-rich membrane regions takes place through protein-protein interactions. However, in rafts this process takes place through interactions between the lipids within the rafts and the transmembrane domain of integral membrane proteins (lipid-protein interaction) or the lipid moiety of proteins attached to the membrane by a lipid modification (lipid-lipid interaction). The recruitment of cytosolic proteins by protein-protein interactions through modular domains (SH2 domains, SH3 domains, etc.) can take place in both raft and non-raft membranes. Proteins excluded from rafts are in yellow; proteins included in rafts are in blue (integral membrane proteins), light brown (GPI-anchored proteins) or pink (acylated, cytosolically-oriented, proteins such as Src family kinases, Ras and heterotrimeric G proteins).

View larger version (31K): [in a new window]   Fig. 2. Lipid raft reorganisation after TCR engagement. At steady state, CD4, Lck, LAT and CD3 are associated with small rafts (red) in T cells. Upon triggering, lipid rafts concentrate in the immunological synapse, gathering together specific membrane proteins. Lck becomes activated and phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3 subunits. These phosphorylated motifs become docking sites for the tandem SH2 domains of the tyrosine kinase ZAP70, which is subsequently activated by tyrosine phosphorylation, probably by Lck. Activated ZAP70 phosphorylates the tyrosine residues present in the transmembrane adapter LAT, which recruits Gads, phospholipase C1 (PLC1) and Grb2. As a consequence, different processes are triggered: (1) LAT-associated Gads bring the adapter protein SLP-76 to the rafts, and this adapter becomes a substrate for ZAP70. Phosphorylated SLP-76 recruits the Tec family protein tyrosine kinase Itk, the guanine-nucleotide-exchange factor Vav and the adapter molecule Nck. Subsequently, Nck recruits the PAK and WASP proteins through its SH3 domains. PAK and WASP are regulated by Vav and in turn regulate the reorganisation of the cytoskeleton. (2) PLC1 recruited to LAT is activated through tyrosine phosphorylation by ZAP70 and Itk. Activated PLC1 converts phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) into diacylglycerol (DAG) and inositol (3,4,5)-trisphosphate (Ins(1,4,5)P3). Subsequently, DAG activates protein kinase C and Ras guanyl-nucleotide-releasing protein (RasGRP), and Ins(1,4,5)P3 activates the transcription factor NF-AT by promoting Ca2+ mobilization and calcineurin activation. (3) Grb2 associated with LAT recruits Sos to the rafts, and this attracts Ras and subsequently other machinery, which results in activation of MAP kinases. Although represented as excluded in resting cells and included in activated cells, the presence in rafts of components of the TCR-CD3 (other than CD3) before and after triggering is controversial (see text). Curved arrows in dark blue indicate relevant tyrosine phosphorylation events occurring upon activation. Horizontal brown arrows indicate tyrosine dephosphorylation events carried out by CD45 molecules present close to the raft edge.

View larger version (40K): [in a new window]   Fig. 3. Transport pathways in polarised epithelial MDCK cells and T lymphocytes. (A) In MDCK epithelial cells, newly synthesised proteins are segregated after passage through the Golgi in different vesicular carriers destined for the apical (red) and basolateral (green) subdomains, which have different protein compositions and functions. Partitioning of proteins into rafts appears to mediate the sorting of at least some apical membrane proteins, such as HA, whereas basolateral sorting (green arrow) is dependent on the existence of a specific signal in the cytoplasmic tail of membrane proteins. Caveolae are raft-containing invaginated structures exclusively located in the basolateral surface. MAL and caveolin-1 are machinery involved in raft-dependent apical transport (straight arrow in red) and caveolae formation (curved arrow in red), respectively. (B) Polarised migrating T lymphocytes display two poles: the leading edge at the front and a membrane protrusion (the uropod) at the trailing edge, each of which has a specific protein composition and function. HA appears to employ rafts for biosynthetic transport (red arrow) to the uropod, which contains rafts. T cells lack caveolin-1 but do express MAL. (C) Caveolin-1 is necessary for caveolae formation and organises lipid rafts to build the caveolar architecture. (D) MAL is necessary for apical transport and appears to organise lipid rafts for the formation of the transport vesicles.

View larger version (18K): [in a new window]   Fig. 4. Vectorial transport of HA in T lymphocytes. Polarised T lymphoblasts infected with the influenza virus were fixed and subjected to double-label immunofluorescence analysis with antibodies specific to HA and to ICAM-3, a uropod protein marker, in the absence of a permeabilization step. The bright field image is depicted to show the cell morphology. The uropod and the direction of migration are indicated by an arrowhead and an arrow, respectively. Controls to assess the specificity of the labelling included incubations with control primary antibodies or omission of the primary antibodies. Bar, 5 µm.