Hai-Tao He

Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains

T‐cell receptors (TCRs) upon binding to peptide–MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (γδϵ) and ζ subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP‐70 kinase and other signaling components. We examined the role of the low‐density detergent‐insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine‐phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM‐associated receptor complexes are highly enriched in the hyperphosphorylated p23 ζ chains, contain most of the TCR/CD3‐associated, phosphorylation‐activated ZAP‐70 kinases and seem to integrate into higher order, multiple tyrosine‐phosphorylated substrate‐containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid‐dependent (microdomain model) or cytoskeleton‐based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin‐based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free‐like diffusion was observed when both the lipid‐dependent and cytoskeleton‐based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

TCR signal initiation machinery is pre‐assembled and activated in a subset of membrane rafts

Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent‐insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37°C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of ∼70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR–CD3 complex, Lck and ZAP‐70 kinases, and CD4 co‐receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

Membrane rafts and signaling by the multichain immune recognition receptors.

The recent recognition of the presence of rafts in the plasma membrane and of their involvement in cell signaling has strongly stimulated the search for their function in receptor-mediated signal transduction in lymphocytes. Recent progress suggests that a general feature of membrane rafts is to serve as platforms wherein the signaling cascades triggered through different multichain immune recognition receptors (e.g. the TCR, BCR and FcepsilonRI) are initiated and organized.

Dynamics in the plasma membrane: how to combine fluidity and order

Cell membranes are fascinating supramolecular aggregates that not only form a barrier between compartments but also harbor many chemical reactions essential to the existence and functioning of a cell. Here, it is proposed to review the molecular dynamics and mosaic organization of the plasma membrane, which are thought to have important functional implications. We will first summarize the basic concepts of Brownian diffusion and lipid domain formation in model membranes and then track the development of ideas and tools in this field, outlining key results obtained on the dynamic processes at work in membrane structure and assembly. We will focus in particular on findings made using fluorescent labeling and imaging procedures to record these dynamic processes. We will also discuss a few examples showing the impact of lateral diffusion on cell signal transduction, and outline some future methodological challenges which must be met before we can answer some of the questions arising in this field of research.

An essential role for membrane rafts in the initiation of Fas/CD95‐triggered cell death in mouse thymocytes

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death‐inducing signaling complex (DISC) that includes Fas, the Fas‐associated death domain protein (FADD) and caspase‐8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid‐ and cholesterol‐rich membrane rafts in mouse thymocytes as well as the L12.10‐Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase‐8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas‐triggered recruitment of FADD and caspase‐8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas‐mediated cell death signaling.

Palmitoylation is required for efficient Fas cell death signaling.

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas‐mediated cell death signaling. Here, we report that the post‐translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell‐free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton‐linked rafts upon Fas stimulation and for the raft‐dependent, ezrin‐mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death‐inducing signaling complex assembly and subsequent caspase cascade leading to cell death.

Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated ζ chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.