Linker For Activation Research Articles

The Requirement of Linker for Activation of T Cells in the Primary and Memory Responses of CD8 T Cells

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that links TCR engagement to downstream signaling events. Although it is clear that LAT is essential in thymocyte development and initiation of T cell activation, its function during T cell expansion, contraction, and memory formation remains unknown. To study the role of TCR-mediated signaling in CD8 T cells during the course of pathogen infection, we used an inducible mouse model to delete LAT in Ag-specific CD8 T cells at different stages of Listeria infection and analyzed the effect of deletion on T cell responses. Our data showed that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction and memory maintenance. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1(low)IL-7R(high)CD62L(high) memory T cells. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Our data demonstrated that, although TCR-mediated signaling is dispensable for contraction and memory maintenance, it regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.

Targeting Galectin-1 Overcomes Breast Cancer-Associated Immunosuppression and Prevents Metastatic Disease

Galectin-1 (Gal1), an evolutionarily conserved glycan-binding protein, contributes to the creation of an immunosuppressed microenvironment at sites of tumor growth. In spite of considerable progress in elucidating its role in tumor-immune escape, the mechanisms underlying the inhibitory functions of Gal1 remain obscure. Here, we investigated the contribution of tumor Gal1 to tumor growth, metastasis, and immunosuppression in breast cancer. We found that the frequency of Gal1(+) cells in human breast cancer biopsies correlated positively with tumor grade, while specimens from patients with benign hyperplasia showed negative or limited Gal1 staining. To examine the pathophysiologic relevance of Gal1 in breast cancer, we used the metastatic mouse mammary tumor 4T1, which expresses and secretes substantial amounts of Gal1. Silencing Gal1 expression in this model induced a marked reduction in both tumor growth and the number of lung metastases. This effect was abrogated when mice were inoculated with wild-type 4T1 tumor cells in their contralateral flank, suggesting involvement of a systemic modulation of the immune response. Gal1 attenuation in 4T1 cells also reduced the frequency of CD4(+)CD25(+) Foxp3(+) regulatory T (T(reg)) cells within the tumor, draining lymph nodes, spleen, and lung metastases. Further, it abrogated the immunosuppressive function of T(reg) cells and selectively lowered the expression of the T-cell regulatory molecule LAT (linker for activation of T cells) on these cells, disarming their suppressive activity. Taken together, our results offer a preclinical proof of concept that therapeutic targeting of Gal1 can overcome breast cancer-associated immunosuppression and can prevent metastatic disease.

Sidewall modification of multiwalled carbon nanotubes by Allivum sativum (garlic) and its effect on the deposition of gold nanoparticles

Sidewall modification of multiwalled carbon nanotubes (abbreviated as MWCNTs) was achieved using Allivum sativum (garlic) extract by an acid-free green process. These organosulfur modified-MWCNTs were then decorated with gold nanoparticles and examined by transmission electron microscopy. The presence of organosulfurs over the modified nanotube surface was confirmed. Nanotube surface modification and subsequent presence of thiols as an active linker was confirmed by Raman spectroscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray and X-ray photoelectron spectroscopy. In the absence of these organosulfurs (thiols), no gold nanoparticle attachment was observed. Both small (1–8nm) and large (12–20nm) gold nanoparticles were found to decorate the modified nanotube surface suggesting coalescence among nanoparticles.

A phospholipase C-γ1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice.

Mice expressing a germline mutation in the phospholipase C-γ1-binding site of linker for activation of T cells (LAT) show progressive lymphoproliferation and ultimately die at 4-6 mo age. The hyperactivated T cells in these mice show defective TCR-induced calcium flux but enhanced Ras/ERK activation, which is critical for disease progression. Despite the loss of LAT-dependent phospholipase C-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major Ras guanine exchange factor responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4(+) T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, protein kinase C-θ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting.

Single Particle Tracking using Fluorogen Activating Peptides to Investigate FcεRI Signaling Dynamics

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils. Cross-linking of IgE-bound FceRI via multivalent antigen initiates a signaling cascade that ultimately results in the release of key mediators for an allergic response. Priming with IgE has been traditionally thought of as a passive event, yet certain forms of IgE termed ‘cytokinergic’ IgEs (cIgE) have been shown to induce activation independent of antigen. The mechanism of action of cIgEs remains unclear. To determine whether changes in receptor mobility are associated with cIgE activation, we have developed a fluorogen activating peptide (FAP)-tagged FceRI ɣ-subunit for single particle tracking. FAPs are genetically expressible tags that bind an exogenous fluorogen dye. The titration of the fluorogen dye allows the labeling density to be adjusted at the time of imaging without the need to photo-bleach or photo-activate a population of dye molecules. We find that even in the presence of concentrations of cIgE that induce robust signaling, FceRI remains highly mobile.We take advantage of the FAP system to additionally investigate the mobility of the downstream scaffolding protein Linker for Activation of T-cells (LAT). The mechanism through which FceRI crosslinking propagates signaling to LAT remains a topic of debate as LAT has been previously shown to form clusters segregated from FceRI after activation. However, these studies were carried out using high concentrations of multivalent allergen that resulted in large immobile FceRI aggregates. The far-red emission of the MG2P fluorogen allows for simultaneous tracking of FAP-LAT mobility with respect to FceRI labeled with Alexa488-IgE. Using FAP-tagged LAT, we investigate how LAT mobility and distribution change over a range of activating conditions, in which FceRI goes from mobile to immobile crosslinked aggregates.

Resolving multi-molecular protein interactions by photoactivated localization microscopy

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.

Assessment of caspase mediated degradation of linker for activation of T cells (LAT) at a single cell level

Caspase/Granzyme B mediated protein degradation is involved in elimination of activated T cell receptor (TCR) signaling molecules during processes of thymocyte selection and maintenance of peripheral homeostasis of T cells. Key components of TCR signaling cassette including LAT undergo biological inactivation in response to pro-apoptotic or anergy inducing environmental stimuli. Although available Western immunoblotting-based techniques are appropriate for detection of protein degradation in bulk populations of target cells, quantitative assessment of this process at a single cell level requires a different approach. Here we report on a novel, flow cytometry-based method for assessment of LAT integrity. This method exploits a loss of an anti-LAT antibody epitope recognition following proteolytic degradation of C-terminal domain of the LAT. We show that the LAT degradation precedes phosphatidylserine translocation to the outer leaflet of the plasma membrane and thus may constitute an early marker of T cell apoptosis. When used in conjunction with multi-parameter flow cytometry, our method revealed that FoxP3+CD4+CD8low thymocytes i.e. precursors of thymus derived CD4+ regulatory T cells, in contrast to Foxp3− CD4+CD8low thymocytes are resistant to LAT degradation in response to CD3ε crosslinking. This finding can be used as an additional marker for T regulatory cell lineage.

Active Belite Cement Clinker Produced with Mineral Waste

This paper presents a laboratory study on active belite cement linker using mineral waste as one of the major raw meal components. The main chemical component of mineral waste employed in this study is silica (SiO2), around 70%. The raw meals were soaked in Muffle Furnace at 1350oC for 10 minutes and 20minutes respectively, then, cooled down to room temperature using air blower. Boron Oxide was used to stabilize high temperature phases of C2S. QXRD analysis indicates that active belite cement clinker has major mineral components consisting of Dicalcium Silicate (C2S), Ferrite (C2 (A0.48F1.52) O5) and trace amount of Tricalcium Silicate (C3S). Among them, Dicalcium silicate is over 85 percent, Ferrite around 10 percent and Tricalcium silicate less than 10 percent. Also, significant amount of high temperature polymorphic C2S was stabilized under room temperature. Among polymorphic belite phases, αH-C2S accounts for around 66% of cement clinker, and αL-C2S for about 22% of cement clinker. Scanning Eαlectronic Microscope (SEM) analysis also shows coαnsiderable round grains of C2S. TGA-DSC spectrum indicated there is no significant phase change while cement clinker was cooling down. Also, the mechanical tests on active belite cement show that active belite cement clinker has a slow strength development at early ages, but rapid strength gain over 70Mpa at 28 days. Thus, this active belite cement clinker demonstrates very promising prospect in sustainable cement industry development.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

DJ-1 regulates mast cell activation and IgE-mediated allergic responses

DJ-1 is an antioxidant protein known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. ROS and DJ-1 levels in serum or culture medium were measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. Patients with atopic dermatitis had increased levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced passive cutaneous anaphylaxis and augmented ROS levels in sera and bone marrow-derived mast cells (BMMCs). Furthermore, antigen-induced degranulation and production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 small interfering RNA-transfected BMMCs compared with that seen in wild-type (WT) BMMCs. Studies with these cells and BMMCs transfected with small interfering RNAs against the phosphatases Src homology domain 2-containing protein tyrosine phosphatase (SHP) 1 and SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity, leading to strengthened signaling through linker for activation of T cells, phospholipase Cγ, and mitogen-activated protein kinases. A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis.

Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation.

It is likely that the enhancement of signaling after antigenic stimulation, particularly in the tumor microenvironment, would improve the function of adoptively transferred T cells. Linker for activation of T cells (LAT) plays a central role in T cell activation. We hypothesized that the ubiquitylation-resistant form of LAT in cells would enhance T cell signaling and thus augment antitumor activity. To test this, human CD4(+) or CD8(+) T cells were electroporated with small interfering RNA (siRNA) to repress endogenous LAT and ubiquitylation-resistant LAT 2KR or wild-type LAT mRNA was introduced for reexpression. Significantly enhanced phosphorylation of LAT and phospholipase C-γ (PLCγ) was observed, and augmented calcium signaling after T cell receptor (TCR) triggering was observed in LAT 2KR-expressing T cells. TCR-induced calcium signaling was abrogated in LAT knockdown cells, but the baseline was higher than that of control siRNA-electroporated cells, suggesting a fundamental requirement of LAT to maintain calcium homeostasis. Redirected LAT 2KR T cells expressing a chimeric antigen receptor or an MHC class I-restricted TCR showed augmented function as assessed by enhanced cytokine secretion and cytotoxicity. These results indicate that interruption of LAT ubiquitylation is a promising strategy to augment effector T cell function for adoptive cell therapy.

The Importance of the Erk Pathway in the Development of Linker for Activation of T Cells-Mediated Autoimmunity

The ability of the transmembrane adaptor protein linker for activation of T cells (LAT) to regulate T cell development, activation, survival, and homeostasis depends upon phosphorylation of its multiple tyrosine residues. The mutation of tyrosine 136 on LAT abrogates its interaction with phospholipase C-γ1, causing severe ramifications on TCR-mediated signaling. Mice harboring this mutation, LATY136F mice, have significantly impaired thymocyte development; however, they rapidly develop a fatal lymphoproliferative disease marked by the uncontrolled expansion of Th2-skewed CD4(+) T cells, high levels of IgE and IgG1, and autoantibody production. In this study, we assessed the contribution of multiple signaling pathways in LATY136F disease development. The deletion of the critical signaling proteins Gads and RasGRP1 caused a further block in thymocyte development, but, over time, could not prevent CD4(+) T cell hyperproliferation. Also, restoring signaling through the NF-κB and NFAT pathways was unable to halt the development of disease. However, expression of a constitutively active Raf transgene enhanced lymphoproliferation, indicating a role for the Ras-MAPK pathway in LAT-mediated disease.

The Adaptor 3BP2 Is Required for Early and Late Events in FcεRI Signaling in Human Mast Cells

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.

Polymorphisms of protein tyrosine phosphatase CD148 influence FcγRIIA-dependent platelet activation and the risk of heparin-induced thrombocytopenia

AbstractHeparin-induced thrombocytopenia (HIT) is due primarily to IgG antibodies specific to platelet factor 4/heparin complexes (PF4/Hs) that activate platelets via FcγRIIA. CD148 is a protein tyrosine phosphatase that regulates Src kinases and collagen-induced platelet activation. Three polymorphisms affecting CD148 (Q276P, R326Q, and D872E) were studied in HIT patients and 2 control groups, with or without antibodies to PF4/Hs. Heterozygote status for CD148 276P or 326Q alleles was less frequent in HIT patients, suggesting a protective effect of these polymorphisms. Aggregation tests performed with collagen, HIT plasma, and monoclonal antibodies cross-linking FcγRIIA showed consistent hyporesponsiveness of platelets expressing the 276P/326Q alleles. In addition, platelets expressing the 276P/326Q alleles exhibited a greater sensitivity to the Src family kinases inhibitor dasatinib in response to collagen or ALB6 cross-linking FcγRIIA receptors. Moreover, the activatory phosphorylation of Src family kinases was considerably delayed as well as the phosphorylation of Linker for activation of T cells and phospholipase Cγ2, 2 major signaling proteins downstream from FcγRIIA. In conclusion, this study shows that CD148 polymorphisms affect platelet activation and probably exert a protec-tive effect on the risk of HIT in patients with antibodies to PF4/Hs.

Tyrosine Phosphorylation-Independent Regulation of Lipopolysaccharide-Mediated Response by the Transmembrane Adaptor Protein LAB

Linker for activation of B cells (LAB)/non-T cell activation linker is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating TCR and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB(-/-) bone marrow-derived dendritic cells. These bone marrow-derived dendritic cells also produced more TNF-α, IL-6, and IL-10 than wild-type cells. Moreover, LAB(-/-) mice were hyperresponsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knockin mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity.

Modeling and Simulation of Aggregation of Membrane Protein LAT with Molecular Variability in the Number of Binding Sites for Cytosolic Grb2-SOS1-Grb2

The linker for activation of T cells (LAT), the linker for activation of B cells (LAB), and the linker for activation of X cells (LAX) form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1∶1 and 2∶1 complexes with the guanine nucleotide exchange factor SOS1. The 2∶1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate). We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

The transmembrane adaptor protein, linker for activation of T cells (LAT), regulates RANKL-induced osteoclast differentiation.

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein we investigate the role of the transmembrane adaptor proteins in RANKL-induced osteoclastogenesis. LAT positively regulates osteoclast differentiation and is up-regulated by RANKL via c-Fos and NFATc1, whereas LAB and LIME act as negative modulators of osteoclastogenesis. In addition, silencing of LAT by RNA interference or overexpression of a LAT dominant negative in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Furthermore, LAT is involved in RANKL-induced PLC(γ) activation and NFATc1 induction. Thus, our data suggest that LAT acts as a positive regulator of RANKL-induced osteoclastogenesis.

The adaptor protein NTAL enhances proximal signaling and potentiates corticosteroid-induced apoptosis in T-ALL

The biology of T-cell acute lymphoblastic leukemia (ALL) is characterized by functional pre-T-cell receptor (TCR) signaling. Non-T-cell activation linker (NTAL) is a nonenzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. In addition, the molecular mechanism of the contribution of NTAL to malignant T-cell ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-cell ALL Jurkat cell line (Jurkat/wt) and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL(+)). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation were analyzed using flow cytometry, Western blot, and quantitative polymerase chain reaction. Jurkat/NTAL(+) cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. In addition, after TCR stimulation, Jurkat/NTAL(+) cells show a higher level of intracellular extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death.

Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution

Lipid microdomains are postulated to regulate many membrane-associated processes but have remained highly controversial. Here we provide the first direct evidence that the plasma membrane of intact, live cells is comprised of a sub-resolution mixture of approximately 76% ordered and 24% disordered lipid domains, which correspond to liquid-ordered and -disordered model membranes. These measurements were based on the unmixing of fluorescence lifetime decays (phasor analysis) obtained from environmentally sensitive membrane dyes that report the degree of lipid packing. Using the transmembrane protein Linker for Activation of T cells (LAT) as an example, we demonstrate that association with ordered domains retarded LAT diffusion and decreased clustering in meso-scaled protein domains as analysed by super-resolution microscopy. Our data therefore propose a membrane model in which the majority of the plasma membrane is covered by cholesterol-dependent, ordered lipid domains that contribute to the non-random distribution and diffusion of membrane constituents.

Transmembrane Adaptor Proteins in the High-Affinity IgE Receptor Signaling

Aggregation of the high-affinity IgE receptor (FcεRI) initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of spleen tyrosine kinase (Syk). Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs). These proteins are characterized by a short extracellular domain, a single transmembrane domain, and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), linker for activation of X cells (LAX), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG), and growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT)]; engagement of four of them (LAT, NTAL, LAX, and PAG) in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production, and chemotaxis.