Project description:The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.
Project description:Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl(-) is bound to HR in a position near the deleted acid. In the HR ground state, Cl(-) binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl(-) from the central T111 site and the tight binding of Cl(-) to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.
Project description:Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
Project description:T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunological synapse (IS). It remains unresolved whether the unique physical structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we created multicomponent protein surfaces presenting lithographically defined patterns of tethered T cell receptor (TCR) ligands (anti-CD3 "activation sites") surrounded by a field of tethered intercellular adhesion molecule-1 (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4(+) T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the physical pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC- clustering in a fraction of cells, and had significantly reduced production of IFN-gamma. These results suggest that focal clustering of TCR ligand characteristic of the "mature" IS may be required under some conditions for full T cell activation.
Project description:Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.
Project description:The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.
Project description:Binding of T cells to antigen-presenting cells leads to the formation of the immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and focused secretion of effector molecules. Here, we show that upon activation of Jurkat cells microtubules project from the MTOC to a ring of the scaffolding protein ADAP, localized at the synapse. Loss of ADAP, but not lymphocyte function-associated antigen 1, leads to a severe defect in MTOC polarization at the immunological synapse. The microtubule motor protein cytoplasmic dynein clusters into a ring at the synapse, colocalizing with the ADAP ring. ADAP coprecipitates with dynein from activated Jurkat cells, and loss of ADAP prevents MTOC translocation and the specific recruitment of dynein to the synapse. These results suggest a mechanism that links signaling through the T cell receptor to translocation of the MTOC, in which the minus end-directed motor cytoplasmic dynein, localized at the synapse through an interaction with ADAP, reels in the MTOC, allowing for directed secretion along the polarized microtubule cytoskeleton.
Project description:We describe an original activity assay for membrane transport that uses the proton motive force-dependent efflux pump MexAB from Pseudomonas aeruginosa. This pump is co-reconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump.
Project description:During antigen recognition by T cells, signaling molecules on the T cell engage ligands on the antigen-presenting cell and organize into spatially distinctive patterns. These are collectively known as the immunological synapse (IS). Causal relationships between large-scale spatial organization and signal transduction have previously been established. Although it is known that receptor transport during IS formation is driven by actin polymerization, the mechanisms by which different proteins become spatially sorted remain unclear. These sorting processes contribute a facet of signal regulation; thus their elucidation is important for ultimately understanding signal transduction through the T cell receptor. Here we investigate protein cluster size as a sorting mechanism using the hybrid live T cell-supported membrane system. The clustering state of the co-stimulatory molecule lymphocyte function-associated antigen-1 (LFA-1) is modulated, either by direct antibody crosslinking or by crosslinking its intercellular adhesion molecule-1 ligand on the supported bilayer. In a mature IS, native LFA-1 generally localizes into a peripheral ring surrounding a central T cell receptor cluster. Higher degrees of LFA-1 clustering, induced by either method, result in progressively more central localization, with the most clustered species fully relocated to the central zone. These results demonstrate that cluster size directly influences protein spatial positioning in the T cell IS. We discuss a sorting mechanism, based on frictional coupling to the actin cytoskeleton, that is consistent with these observations and is, in principle, extendable to all cell surface proteins in the synapse.
Project description:The interaction between T cells and APCs bearing cognate antigen results in the formation of an immunological synapse (IS). During this process, many receptors and signaling proteins segregate to regions proximal to the synapse. This protein movement is thought to influence T cell function. However, some proteins are transported away from the IS, which is controlled in part by ERM family proteins. Tim-1 is a transmembrane protein with co-stimulatory functions that is found on many immune cells, including T cells. However, the expression pattern of Tim-1 on T cells upon activation by APCs has not been explored. Interestingly, in this study we demonstrate that the majority of Tim-1 on activated T cells is excluded from the IS. Tim-1 predominantly resides outside of the IS, and structure/function studies indicate that the cytoplasmic tail influences Tim-1 polarization. Specifically, a putative ERM binding motif (KRK 244-246) in the Tim-1 cytoplasmic tail appears necessary for proper Tim-1 localization. Furthermore, mutation of the KRK motif results in enhanced early tyrosine phosphorylation downstream of TCR/CD28 stimulation upon ectopic expression of Tim-1. Paradoxically however, the KRK motif is necessary for Tim-1 co-stimulation of NFAT/AP-1 activation and co-stimulation of cytokine production. This work reveals unexpected complexity underlying Tim-1 localization and suggests potentially novel mechanisms by which Tim-1 modulates T cell activity.