Cell Receptor Microclusters Research Articles

DRAK2 regulates myosin light chain phosphorylation in T cells.

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2 or STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear. Gaining further insight into the function of DRAK2 in T cells will shed light on differentially regulated pathways in autoreactive and pathogen-specific T cells, which is critical for improving autoimmune therapies. Here, we demonstrate that DRAK2 contributes to activation of myosin light chain (MLC) in both murine and human T cells. In the absence of Drak2, the amount of polymerized actin was decreased, suggesting that DRAK2 modulates actomyosin dynamics. We further show that myosin-dependent T cell functions, such as migration, T cell receptor microcluster accumulation, and conjugation to antigen presenting cells are decreased in the absence of Drak2. These findings reveal that DRAK2 plays an important role in regulating MLC activation within T cells.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Cytotoxic Tcells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.

PI3K- and PTEN-Governed PIP<sub>2</sub> and PIP<sub>3</sub> Equilibrium Regulates the Activation and Function of B Cells Through Dock2 Controlled Growth of Antigen Receptor Microclusters

The growth of B cell receptor (BCR) microclusters upon antigen stimulation is one of the most important events in B cell activation, however the underlying molecular mechanism unknown. Here, we systematically addressed this question through a combination of molecular imaging, genetic ablation, pharmacological inhibitor and mice model approaches. We showed that the earliest BCR signaling molecules, Lyn, Syk and PLCγ2 are required for B cells to initiate the growth of BCR microclusters. PI3K-mediated PIP3 production but not Akt and PDK1 activation was required for the sustained growth of the BCR microclusters. The PI3K counterpart signaling molecule, PTEN, potently inhibited the growth of the BCR microclusters, which was dependent on its membrane binding activities and on the lipid phosphatase but not protein phosphatase activity. Mechanistically, we showed that PIP3-dependent recruitment and activation of a unique guanine nucleotide exchange factor, Dock2, was required for the sustained growth of the BCR microclusters through the remodeling of the F-actin cytoskeleton. As a consequence, the formation of the antigen-induced B cell immunological synapse (IS) in Dock2 deficient B cells was drastically impaired. Moreover, Dock2 deficient B cells exhibited decreased Ca2 influx. Physiologically, we found that mice with reduced expression of PTEN in B cells showed enhanced antibody responses and a spontaneous lupus-like autoimmune phenotype. Clinically, we showed that primary B cells from lupus patients exhibited more prominent BCR and PI3K microclusters than B cells from healthy controls. Thus, these results demonstrated the importance of PI3K- and PTEN-governed PIP2 and PIP3 equilibrium in regulating the dynamic growth of BCR microclusters during the initiation of B cell activation.

Growth of B Cell Receptor Microclusters Is Regulated by PIP2 and PIP3 Equilibrium and Dock2 Recruitment and Activation

The growth of B cell receptor (BCR) microclusters upon antigen stimulation drives B cell activation. Here, we show that PI3K-mediated PIP3 production is required for the growth of BCR microclusters. This growth is likely inhibited by PTEN and dependent on its plasma membrane binding and lipid phosphatase activities. Mechanistically, we find that PIP3-dependent recruitment and activation of a guanine nucleotide exchange factor, Dock2, is required for the sustained growth of BCR microclusters through remodeling of the F-actin cytoskeleton. As a consequence, Dock2 deficiency significantly disrupts the structure of the B cell immunological synapse. Finally, we find that primary B cells from systemic lupus erythematosus (SLE) patients exhibit more prominent BCR and PI3K microclusters than B cells from healthy controls. These results demonstrate the importance of a PI3K- and PTEN-governed PIP2 and PIP3 equilibrium in regulating the activation of B cells through Dock2-controlled growth of BCR microclusters.

Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner.

Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS.

Multi-color single-molecule tracking and subtrajectory analysis for quantification of spatiotemporal dynamics and kinetics upon T cell activation

The dynamic properties of molecules in living cells are attracting increasing interest. We propose a new method, moving subtrajectory analysis using single-molecule tracking, and demonstrate its utility in the spatiotemporal quantification of not only dynamics but also the kinetics of interactions using single-color images. Combining this technique with three-color simultaneous single-molecule imaging, we quantified the dynamics and kinetics of molecules in spatial relation to T cell receptor (TCR) microclusters, which trigger TCR signaling. CD3ε, a component of the TCR/CD3 complex, and CD45, a phosphatase positively and negatively regulating signaling, were each found in two mobility states: faster (associated) and slower (dissociated) states. Dynamics analysis suggests that the microclusters are loosely composed of heterogeneous nanoregions, possibly surrounded by a weak barrier. Kinetics analysis quantified the association and dissociation rates of interactions with the microclusters. The associations of both CD3ε and CD45 were single-step processes. In contrast, their dissociations were each composed of two components, indicating transient and stable associated states. Inside the microclusters, the association was accelerated, and the stable association was increased. Only CD45 showed acceleration of association at the microcluster boundary, suggesting specific affinity on the boundary. Thus, this method is an innovative and versatile tool for spatiotemporal quantification.

Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

Correction: Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse.

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation.

The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

Human tonsil naive B cells and germinal center B cells show distinct behaviors upon contact of antigen in lipid bilayers

Abstract During antibody responses to T-dependent antigens B cells undergo clonal expansion and somatic hyper-mutation in the dark zones of germinal centers (GCs) and subsequently undergo isotype switching and affinity based selection in the GC light zones. Only a small portion of B cells survive from GC reactions and differentiate into either plasma cells or memory B cells. However, the mechanism underlying selection within the GC is not fully understood. We compared the behavior of human tonsil naïve (IgD+, CD10−) and GC (IgD−, CD10+) B cells as they engaged antigen (using Light chain specific antibodies as a surrogate antigen) incorporated into planar lipid bilayers using a combination of TIRF and confocal microscopy. We observed marked differences in the cellular dynamics of naïve and GC B cells. Upon touching the antigen-containing bilayer naïve B cells rapidly form B cell receptor (BCR) micro-clusters that grow rapidly with time and signal for Ca++ fluxes. Within five minutes naïve B cells formed stable areas of contact with the bilayers. In contrast, GC B cells only slowly form BCR micro-clusters that grow slowly with time and signal for delayed Ca++ fluxes. Notably, the behavior of GC B cells over the same time period is far more dynamic with only transient interactions with the bilayer and active ruffling in the cells periphery suggesting that the GC B cells in contrast to naïve B cells continue to sample antigen for longer periods of time. Experiments in progress are aimed at relating the observed behaviors to the affinity of naïve and GC B cells for antigen.

A negative-feedback function of PKCβ in the formation and accumulation of signaling-active B cell receptor microclusters within B cell immunological synapse.

Advanced live cell imaging studies suggested that B cell activation is initiated by the formation of BCR microclusters and subsequent B cell IS upon BCR and antigen recognition. PKC family member PKCβ is highly expressed in B cells and plays an important role in the initiation of B cell activation. Here, we reported an inhibitory function of PKCβ through a negative-feedback manner in B cell activation. Compared with WT (PKCβ-WT) or the constitutively active (PKCβ-ΔNPS) form of PKCβ, DN PKCβ (PKCβ-DN) unexpectedly enhanced the accumulation of BCR microclusters into the B cell IS, leading to the recruitment of an excessive amount of pSyk, pPLC-γ2, and pBLNK signaling molecules into the membrane-proximal BCR signalosome. Enhanced calcium mobilization responses in the decay phase were also observed in B cells expressing PKCβ-DN. Mechanistic studies showed that this negative-feedback function of PKCβ works through the induction of an inhibitory form of pBtk at S180 (pBtk-S180). Indeed, the capability of inducing the formation of an inhibitory pBtk-S180 is in the order of PKCβ-ΔNPS > PKCβ-WT > PKCβ-DN. Thus, these results improve our comprehensive understanding on the positive and negative function of PKCβ in the fine tune of B cell activation.

Dynamic Behavior of TCR Microclusters on a Live Cell Investigated by a Gold Nanoparticle Array

T cell receptor (TCR) microclusters play an important role as they are the sites of regulation of early T cell signaling. TCR microclusters are, however, difficult to study because their size is too small for conventional fluorescence microscopy and too large for physicochemical methods that depend on proximity effect such as FRET. Despite the recent advancement in superresolution microscopies, their use is still limited to fixed cells or slow processes in the cell. We developed a new platform for probing and manipulating TCR microclusters on a live cell by mechanical means.

Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

Local changes in lipid environment of T cell receptor microclusters regulate membrane binding by the CD3epsilon cytoplasmic domain (P1103)

Abstract The CD3epsilon and zeta cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane, and a previous NMR structure showed that both tyrosines of the CD3epsilon Immuno-tyrosine based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3epsilon residues were previously shown to be essential for CD3epsilon and zeta membrane binding. Phosphatidylserine is the most abundant negatively charged lipid on the inner leaflet of the plasma membrane and makes a major contribution to membrane binding by the CD3epsilon cytoplasmic domain. Here we show that T cell receptor triggering by peptide-MHC complexes induces dissociation of the CD3epsilon cytoplasmic domain from the plasma membrane. Release of the CD3epsilon cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and phosphatidylserine density in T cell receptor microclusters. The localized reduction in phosphatidylserine density is explained by reduced diffusion of this lipid into synapses. These changes in the lipid composition of T cell receptor microclusters even occur when receptor signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of the microclusters thus render the CD3epsilon cytoplasmic domain accessible during early stages of T cell activation.

T Cell Receptor-Associated Actin Patches Generated by Wiskott-Aldrich Syndrom Protein

T cell activation involves massive cytoskeletal remodeling, including dynamic actin polymerization and centripetal flow proximal to T cell-antigen presenting surface synapse (IS). F-actin is required for the formation of signaling T cell receptor microclusters (MCs) and for on-going signaling. Wiskott-Aldrich Syndrome Protein (WASP) is an actin nucleation-promoting factor that is critical for appropriate T cell function. Patients with mutations in WASP experience a severe immunodeficiency and autoimmunity. The role of WASP in actin polymerization at the immunological synapse has been surprisingly cryptic as WASP deficient T cells still display dramatic increases in F-actin at the immunological synapse. Here, with the aid of live cell as well as super-resolution imaging in primary T cells, we identify WASP dependent F-actin patches closely associated with TCR microclusters. The patches also contain phosphorylated HS-1, a cortactin homolog, and phosphorylated Cas-L a mechanotransduction protein, which are recruited down-stream of WASP. Closely related proteins N-WASP and WAVE2 do not participate in TCR-associated F-actin patches. Super-resolution imaging reveals that the patches are composed of multiple actin arrays that are oriented to diverge from TCR microclusters. We conclude that F-actin patches represent a unique mode of actin polymerization in the immunological synapse that integrate the well documented functional defects in WASP deficient T cells to F-actin nucleation activity.

Investigating the Dynamic Behavior of TCR Microclusters by a Gold Nanoparticle Array

T cell receptor (TCR) microclusters play an important role in early signaling in T cells, and their properties and dynamic behavior are the active target of research. TCR microclusters are, however, difficult to study because their size is too small for conventional fluorescence microscopy and too large for physicochemical methods that depend on proximity effect such as FRET. We developed a new platform for probing and manipulating TCR microclusters by mechanical means. A two-dimensional array of gold nanoparticles is embedded on the glass substrate. A supported membrane, which acts as a surrogate cell membrane that has laterally fluid ligand molecules for cells, was deposited on a glass substrate with a regular array of gold nanoparticles. The supported membrane interacted with T cells and the behavior of TCR microclusters on the T cell surface was effectively altered by the gold nanoparticles. This platform offers many opportunities for studying this poorly understood size regime of protein assemblies.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Microfabricated platforms to modulate and monitor T cell synapse assembly

Immunological synapse (IS) is a complex supramolecular structure formed at the interface between T cells and antigen presenting cells (APCs) during T cell antigen recognition. Microfabricated platforms have made great contributions to our understanding of the assembly dynamics and functional roles of the T cell synapses over the last decade. Here, we review three different types of microfabricated platforms developed to modulate and monitor the T cell synapse assembly. Firstly, multi-protein micropatterned surfaces presenting key ligands for T cell activation that can modulate the spatial distribution of receptors/signaling molecules in T cell synapses are described. Secondly, micropatterned supported bilayers that can modulate the dynamics of T cell receptor (TCR) microclusters are introduced. Lastly, T-APC pair arrays that allow for improved fluorescence live cell imaging are discussed.

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell's encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.