A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Opto-APC: Engineering of cells that display phytochrome B on their surface for optogenetic studies of cell-cell interactions.

The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks.

Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates in vivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis.

Septins organize endoplasmic reticulum-plasma membrane junctions for STIM1-ORAI1 calcium signalling.

ORAI1 Ca2+ channels in the plasma membrane (PM) are gated by STIM1 at endoplasmic reticulum (ER)-PM junctions to effect store-dependent Ca2+ entry into cells, but little is known about how local STIM-ORAI signalling at junctions is coordinated with overall cellular architecture. Filamentous septins can specify cytoskeletal rearrangements and have been found recently to modulate STIM-ORAI signalling. Here we show by super-resolution imaging of ORAI1, STIM1, and septin 4 in living cells that septins facilitate Ca2+ signalling indirectly. Septin 4 does not colocalize preferentially with ORAI1 in resting or stimulated cells, assemble stably at ER-PM junctions, or specify a boundary that directs or confines ORAI1 to junctions. Rather, ORAI1 is recruited to junctions solely through interaction with STIM proteins, while septins regulate the number of ER-PM junctions and enhance STIM1-ORAI1 interactions within junctions. Thus septins communicate with STIM1 and ORAI1 through protein or lipid intermediaries, and are favorably positioned to coordinate Ca2+ signalling with rearrangements in cellular architecture.

Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling.

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)-binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθHR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and TH2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr126 of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr315 and Tyr319 were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθHR2A-expressing CD4+ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and TH2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca2+ signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.

A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.

Cell-surface-receptor pathways amplify weak, rare and local stimuli to induce cellular responses. This task is accomplished despite signaling components that segregate into nanometer-scale membrane domains. Here we describe a 'catch-and-release' mechanism that amplified and dispersed stimuli by releasing activated kinases from receptors lacking intrinsic catalytic activity. Specifically, we discovered a cycle of recruitment, activation and release for Zap70 kinases at phosphorylated T cell antigen receptors (TCRs). This turned the TCR into a 'catalytic unit' that amplified antigenic stimuli. Zap70 released from the TCR remained at the membrane, translocated, and phosphorylated spatially distinct substrates. The mechanisms described here are based on widely used protein domains and post-translational modifications; therefore, many membrane-associated pathways might employ similar mechanisms for signal amplification and dispersion.

Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes.

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation.

The B cell antigen receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. Mature B cells have both immunoglobulin M (IgM) and IgD types of BCRs, which have identical antigen-binding sites and are both associated with the signaling subunits Igα and Igß, but differ in their membrane-bound heavy chain isoforms. By two-color direct stochastic optical reconstruction microscopy (dSTORM), we showed that IgM-BCRs and IgD-BCRs reside in the plasma membrane in different protein islands with average sizes of 150 and 240 nm, respectively. Upon B cell activation, the BCR protein islands became smaller and more dispersed such that the IgM-BCRs and IgD-BCRs were found in close proximity to each other. Moreover, specific stimulation of one class of BCR had minimal effects on the organization of the other. These conclusions were supported by the findings from two-marker transmission electron microscopy and proximity ligation assays. Together, these data provide evidence for a preformed multimeric organization of BCRs on the plasma membrane that is remodeled after B cell activation.

T cell receptor dwell times control the kinase activity of Zap70.

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity.

FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck.

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

Super-resolution microscopy: a comparative treatment.

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.

How membrane structures control T cell signaling.

Genetic and biochemical studies have identified a large number of molecules involved in T cell signaling. They have provided us with a comprehensive understanding of protein-protein interactions and protein modifications that take place upon antigen recognition. Diffraction limited fluorescence microscopy has been used to study the distribution of signaling molecules on a cellular level. Specifically, the discovery of microclusters and the immunological synapse demonstrates that T cell signaling cascades utilizes spatial association and segregation. Recent advancements in live cell imaging have allowed us to visualize the spatio-temporal mechanisms of T cell signaling at nanometer scale resolution. This led to the discovery that proteins are organized in distinct membrane domains prior and during T cell activation. Evidently, plasma membrane structures and signaling molecule distributions at all length scales (molecular to cellular) are intrinsic to the mechanisms that govern signaling initiation, transduction, and inhibition. Here we provide an overview of possible plasma membrane models, molecular assemblies that have been described to date, how they can be visualized and how they might contribute to T cell signaling.

Fluorescence correlation spectroscopy: linking molecular dynamics to biological function in vitro and in situ.

Fluorescence correlation spectroscopy (FCS) is a minimally invasive real-time fluorescence technique capable of detecting single molecules in vitro and in situ. By recording and correlating the fluctuations in fluorescence intensity measurements, it is possible to obtain information on molecular mobility and diffusion, hydrodynamic radii, local concentrations and photochemical and photophysical properties. By using dual-color cross-correlation spectroscopy, it is possible to monitor highly specific molecular-level interactions such as binding processes and chemical reactions. Recent advances in alternative detection schemes have allowed the extension of these techniques to the monitoring of slower timescales (e.g. Raster Image Correlation Spectroscopy-RICS) or higher concentrations (e.g. Total Internal Reflection-TIR-FCS). Given the versatility of these techniques, they have become commonplace tools used to specifically unravel the spatio-temporal dynamics of macromolecular entities in living biological systems.

Probing the plasma membrane structure of immune cells through the analysis of membrane sheets by electron microscopy.

This chapter describes a method to generate plasma membrane sheets that are large enough to visualize the membrane architecture and perform quantitative analyses of protein distributions. This procedure places the sheets on electron microscopy grids, parallel to the imaging plane of the microscope, where they can be characterized by transmission electron microscopy. The basic principle of the technique is that cells are broken open ("ripped") through mechanical forces applied by the separation of two opposing surfaces sandwiching the cell, with one of the surfaces coated onto an EM grid. The exposed inner membrane surfaces can then be visualized with electron dense stains and specific proteins can be detected with gold conjugated probes.

Evidence for a functional sidedness to the alphabetaTCR.

The T cell receptor (TCR) and associated CD3gammaepsilon, deltaepsilon, and zetazeta signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the alphabeta and gammadeltaTCR-CD3 complexes. We also report that the other side of the alphabetaTCR mediates homotypic alphabetaTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3epsilon chains of two CD3 heterodimers are arranged side-by-side in both the alphabeta and gammadeltaTCR-CD3 complexes. This system was also used to show that alphabetaTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Calpha domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair alphabetaTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a "functional-sidedness" to the alphabetaTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located.

TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide-major histocompatibility complex (pMHC) molecules on other cells. If productive, these interactions promote the formation of an immunological synapse. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR-pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4-12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR-pMHC complexes, indicating that the TCR binds pMHC independently of CD4.

TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.