RESUMO
T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell-nanopatterned supported lipid bilayer system to quantitatively probe the actin-TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.
Assuntos
Actinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Citoesqueleto de Actina/metabolismo , Algoritmos , Animais , Células Cultivadas , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/ultraestrutura , Ativação Linfocitária , Camundongos , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/química , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.
Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Biofísica/métodos , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Sinalização do Cálcio , Sistema Imunitário , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Bicamadas Lipídicas/química , Lipídeos/química , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , CamundongosRESUMO
The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor. Although this approach has yielded insights into EphA2 signaling, preclustered ligands bypass natural receptor clustering processes and thus mask any role of clustering as a signal regulatory mechanism. Here, we present EphA2-expressing cells with a fusion protein of monomeric ephrin-A1 (mEA1) and enhanced monomeric yellow fluorescent protein that is linked to a supported lipid bilayer via a nickel-decahistidine anchor. The mEA1 is homogeneously dispersed, laterally mobile, and monomeric as measured by fluorescence imaging, correlation spectroscopy, and photon counting histogram analysis, respectively. Ephrin-A1 presented in this manner activates EphA2 on the surface of MDA-MB-231 human breast cancer cells, as measured by EphA2 phosphorylation and degradation. Spatial mutation experiments in which nanopatterns on the underlying substrate restrict mEA1 movement in the supported lipid bilayer reveal spatio-mechanical regulation of this signaling pathway, consistent with recently reported observations using a synthetically cross-linked ephrin-A1 dimer.
Assuntos
Efrina-A1/metabolismo , Bicamadas Lipídicas/metabolismo , Receptor EphA2/metabolismo , Proteínas de Bactérias/metabolismo , Western Blotting , Extratos Celulares , Linhagem Celular Tumoral , Ativação Enzimática , Imunofluorescência , Humanos , Proteínas Luminescentes/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Propriedades de SuperfícieRESUMO
Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/agonistas , Proteína Son Of Sevenless de Drosófila/química , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Humanos , Cinética , Nucleotídeos/química , Proteína Son Of Sevenless de Drosófila/genéticaRESUMO
Physical inputs, both internal and external to a cell, can directly alter the spatial organization of cell surface receptors and their associated functions. Here we describe a protocol that combines solid-state nanolithography and supported lipid membrane techniques to trigger and manipulate specific receptors on the surface of living cells and to develop an understanding of the interplay between spatial organization and receptor function. While existing protein-patterning techniques are capable of presenting cells with well-defined clusters of protein, this protocol uniquely allows for the control of the spatial organization of laterally fluid receptor-ligand complex at an intermembrane junction. A combination of immunofluorescence and single-cell microscopy methods and complementary biochemical analyses are used to characterize receptor signaling pathways and cell functions. The protocol requires 2-5 d to complete depending on the parameters to be studied. In principle, this protocol is widely applicable to eukaryotic cells and herein is specifically developed to study the role of physical organization and translocation of the EphA2 receptor tyrosine kinase across a library of model breast cancer cell lines.
Assuntos
Membrana Celular/metabolismo , Membranas Artificiais , Receptor EphA2/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Biotinilação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Feminino , Recuperação de Fluorescência Após Fotodegradação , Humanos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Transporte Proteico , Receptores de Superfície Celular/análise , Coloração e RotulagemRESUMO
Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.