RESUMO
TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.
Assuntos
Antígenos de Histocompatibilidade Classe I/fisiologia , Ativação Linfocitária/fisiologia , Adulto , Feminino , Humanos , Cinética , Ligantes , Complexo Principal de Histocompatibilidade/fisiologia , Masculino , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Oligopeptídeos , Peptídeos , Ligação Proteica/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Imagem Individual de Molécula , Linfócitos T/fisiologiaRESUMO
How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.
Assuntos
Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/química , Transdução de Sinais , Animais , Células COS , Membrana Celular/química , Chlorocebus aethiops , Dimerização , Receptores ErbB/metabolismo , Humanos , Modelos MolecularesRESUMO
Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.
Assuntos
Fragmentos Fab das Imunoglobulinas , Transdução de Sinais , Linfócitos T , Humanos , Cinética , Ligantes , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , DNA/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Ativação Linfocitária , Mutação PuntualRESUMO
Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin-binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the â¼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.
Assuntos
Receptores ErbB , Transdução de Sinais , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/metabolismo , Fosforilação , Fosfotirosina/metabolismoRESUMO
Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation.
Assuntos
Retroalimentação Fisiológica , Proteínas ras , Proteínas ras/metabolismo , Proteínas ras/química , Cinética , Regulação Alostérica , Proteína SOS1/metabolismo , Proteína SOS1/química , Proteína SOS1/genética , Ativação Enzimática , Membrana Celular/metabolismo , Proteínas Son Of Sevenless/metabolismo , Proteínas Son Of Sevenless/química , HumanosRESUMO
Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor (GEF) that plays a central role in numerous cellular signaling pathways. Like many other signaling molecules, SOS is autoinhibited in the cytosol and activates only after recruitment to the membrane. The mean activation time of individual SOS molecules has recently been measured to be â¼60 s, which is unexpectedly long and seemingly contradictory with cellular signaling timescales, which have been measured to be as fast as several seconds. Here, we rectify this discrepancy using a first-passage time analysis to reconstruct the effective signaling timescale of multiple SOS molecules from their single-molecule activation kinetics. Along with corresponding experimental measurements, this analysis reveals how the functional response time, comprised of many slowly activating molecules, can become substantially faster than the average molecular kinetics. This consequence stems from the enzymatic processivity of SOS in a highly out-of-equilibrium reaction cycle during receptor triggering. Ultimately, rare, early activation events dominate the macroscopic reaction dynamics.
Assuntos
Modelos Químicos , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Ativação Enzimática , Cinética , Imagem Individual de MoléculaRESUMO
Here, we present detailed kinetic analyses of a panel of soluble lipid kinases and phosphatases, as well as Ras activating proteins, acting on their respective membrane surface substrates. The results reveal that the mean catalytic rate of such interfacial enzymes can exhibit a strong dependence on the size of the reaction system-in this case membrane area. Experimental measurements and kinetic modeling reveal how stochastic effects stemming from low molecular copy numbers of the enzymes alter reaction kinetics based on mechanistic characteristics of the enzyme, such as positive feedback. For the competitive enzymatic cycles studied here, the final product-consisting of a specific lipid composition or Ras activity state-depends on the size of the reaction system. Furthermore, we demonstrate how these reaction size dependencies can be controlled by engineering feedback mechanisms into the enzymes.
Assuntos
Tamanho Celular , Enzimas/metabolismo , Membranas/fisiologia , Retroalimentação , Cinética , Bicamadas Lipídicas , Lipídeos , Modelos Biológicos , Monoéster Fosfórico Hidrolases , Transdução de SinaisRESUMO
Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.
Assuntos
Nanoporos , Proteínas/metabolismo , Imagem Individual de Molécula , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Camundongos , Linfócitos T/metabolismoRESUMO
Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras)/química , Quinases raf/química , Galectinas/química , Galectinas/genética , Galectinas/metabolismo , Humanos , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/genética , Quinases raf/metabolismoRESUMO
Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.
Assuntos
Fotossíntese , Proteínas , Transferência de Energia , Bicamadas Lipídicas/químicaRESUMO
T cells exhibit remarkable sensitivity and selectivity in detecting and responding to agonist peptides (p) bound to MHC molecules in a sea of self pMHC molecules. Despite much work, understanding of the underlying mechanisms of distinguishing such ligands remains incomplete. Here, we quantify T cell discriminatory capacity using channel capacity, a direct measure of the signaling network's ability to discriminate between antigen-presenting cells (APCs) displaying either self ligands or a mixture of self and agonist ligands. This metric shows how differences in information content between these two types of peptidomes are decoded by the topology and rates of kinetic proofreading signaling steps inside T cells. Using channel capacity, we constructed numerically substantiated hypotheses to explain the discriminatory role of a recently identified slow LAT Y132 phosphorylation step. Our results revealed that in addition to the number and kinetics of sequential signaling steps, a key determinant of discriminatory capability is spatial localization of a minimum number of these steps to the engaged TCR. Biochemical and imaging experiments support these findings. Our results also reveal the discriminatory role of early negative feedback and necessary amplification conferred by late positive feedback.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/imunologia , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Humanos , Células Jurkat , Cinética , Ligantes , Modelos Teóricos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.
Assuntos
Efrina-A1 , Receptor EphA2 , Fator de Crescimento Epidérmico , Receptores ErbB/metabolismo , Retroalimentação , Receptor EphA2/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Membrana Celular/ultraestrutura , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Ativação Linfocitária/imunologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/genética , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Modelos Biológicos , Fosfoproteínas/genética , Transporte Proteico , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/ultraestrutura , TransfecçãoRESUMO
The coordinated organization of cell membrane receptors into diverse micrometre-scale spatial patterns is emerging as an important theme of intercellular signalling, as exemplified by immunological synapses. Key characteristics of these patterns are that they transcend direct protein-protein interactions, emerge transiently and modulate signal transduction. Such cooperativity over multiple length scales presents new and intriguing challenges for the study and ultimate understanding of cellular signalling. As a result, new experimental strategies have emerged to manipulate the spatial organization of molecules inside living cells. The resulting spatial mutations yield insights into the interweaving of the spatial, mechanical and chemical aspects of intercellular signalling.
Assuntos
Junções Intercelulares/metabolismo , Transdução de Sinais , Animais , Humanos , Sinapses Imunológicas/metabolismoRESUMO
The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on protein-ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton's tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a phosphatidylinositol (3-5)-trisphosphate (PIP3) sensing mechanism that achieves switch-like sensitivity to PIP3 levels, surpassing the intrinsic affinity discrimination of PIP3:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Studies in live cells confirm that mutations at the dimer interface and peripheral site produce effects comparable to that of the kinase-dead Btk in vivo. These results demonstrate how a single protein module can institute an allosteric counting mechanism to achieve high-precision discrimination of ligand concentration. Furthermore, this activation mechanism distinguishes Btk from other Tec family member kinases, Tec and Itk, which we show are not capable of dimerization through their PH-TH modules. This suggests that Btk plays a critical role in the stringency of the B cell response, whereas T cells rely on other mechanisms to achieve stringency.
Assuntos
Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Transdução de Sinais/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Galinhas , Camundongos , Modelos Moleculares , Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Conformação Proteica , Domínios Proteicos/fisiologia , Multimerização ProteicaRESUMO
Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.
Assuntos
Proteínas de Bactérias/química , Fatores de Troca do Nucleotídeo Guanina/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/química , Monoéster Fosfórico Hidrolases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cinética , Legionella pneumophila/química , Legionella pneumophila/enzimologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipase C delta/química , Fosfolipase C delta/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Processos Estocásticos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRß Fab' antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab'-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab'-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab'-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.
Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Membrana Celular/metabolismo , Epitopos , Humanos , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.
Assuntos
Lipídeos de Membrana , Proteínas de Membrana , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação , Fosfotirosina , Proteínas Son Of Sevenless/metabolismoRESUMO
Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.