James H. Felce

TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis.

Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn’s disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.

The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A: IMPLICATIONS FOR LIGAND BINDING AND SIGNALING*

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.

Super-Resolved Traction Force Microscopy (STFM)

Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.

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

Activating T cells reorganize their cortical actin to form a ramified transportation network beneath the immunological synapse. 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.

Self-organizing actin patterns shape membrane architecture but not cell mechanics.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.

Type-3 BRET, an Improved Competition-Based Bioluminescence Resonance Energy Transfer Assay

We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.

Unraveling receptor stoichiometry using bret.

The first and arguably most important question that could be asked about the biology of any protein is: does it function alone? Cell surface receptors present special problems for stoichiometric analysis because, being located within lipid bilayers, they are often very hydrophobic, which means that once isolated they can exhibit a strong tendency to aggregate. A very welcome development, therefore, has been the advent of in situ methods for probing receptor organization, the most important of which are presently based on resonance energy transfer. Our first bioluminescence resonance energy transfer (BRET) experiments were, however, inconclusive since both monomeric and dimeric receptors gave high levels of energy transfer (James et al., 2006). It was only with the application of theoretical principles first developed for (Fung and Stryer, 1978; Wolber and Hudson, 1979), and then used in (Kenworthy and Edidin, 1998), Forster resonance energy transfer (FRET) experiments that we could use BRET to confidently distinguish between monomers and dimers. n nWe were very keen to test G protein-coupled receptors (GPCRs) using the new approach given the great interest in these important proteins forming constitutive oligomeric complexes (Angers et al., 2000; Ramsay et al., 2002; Babcock et al., 2003). This seemed unlikely to us firstly because, structurally, GPCRs are ideally configured for functioning autonomously (Meng and Bourne, 2001) and, secondly, because functional autonomy explains the remarkable evolutionary success (Schioth and Fredriksson, 2005) of this very large family of receptors. We were initially ignorant of the extent to which BRET was used to buttress the “GPCRs as oligomers” concept (Pfleger and Eidne, 2005), but when our initial analyses of human β2-adrenergic (β2AR) and mouse cannabinoid (mCannR2) receptors yielded the “BRET signatures” of monomers (James et al., 2006), we had to confront this body of data. The resulting controversy (Bouvier et al., 2007; James and Davis, 2007a,b; Salahpour and Masri, 2007) seems to have prompted the development of other, more complicated approaches. Here, we describe our experiences using BRET and briefly consider the merits of these alternative approaches.

Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages.

Signal integration between activating Fc receptors and inhibitory signal regulatory protein &agr; (SIRP&agr;) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fc&ggr; receptor I (Fc&ggr;RI), Fc&ggr;RII, and SIRP&agr; are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of Fc&ggr;RI, but not Fc&ggr;RII, are constitutively associated with nanoclusters of SIRP&agr;, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of Fc&ggr;RI and SIRP&agr; nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRP&agr; with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, Fc&ggr;RI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration.