title: Multimolecular analysis of stable immunological synapses reveals sustained recruitment and sequential assembly of signaling clusters.
abstract: The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3ζ, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLCγ1 recruitment, pSLP76, and increased CXCR4. Together, these data demonstrate the use of multi-epitope ligand cartography to quantitatively analyze synapse formation and reveal successive recruitment of structural and signaling proteins and sustained phosphorylation at the mature synapse.
authors.affiliation: Otto von Guericke University, Institute of Molecular and Clinical Immunology, Leipziger Str. 44, 39120 Magdeburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Philipsen
authors.forename: Thomas
authors.initials: T
authors.lastname: Engels
authors.forename: Kerstin
authors.initials: K
authors.lastname: Schilling
authors.forename: Slavyana
authors.initials: S
authors.lastname: Gurbiel
authors.forename: Klaus-Dieter
authors.initials: KD
authors.lastname: Fischer
authors.forename: Kerry
authors.initials: K
authors.lastname: Tedford
authors.forename: Burkhart
authors.initials: B
authors.lastname: Schraven
authors.forename: Matthias
authors.initials: M
authors.lastname: Gunzer
authors.forename: Peter
authors.initials: P
authors.lastname: Reichardt
mesh_terms: D000818:Animals; D002645:Chickens; D016000:Cluster Analysis; D018604:Epitope Mapping; D000939:Epitopes / Q000378:metabolism; D054992:Immunological Synapses / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D051567:Proto-Oncogene Proteins c-vav / Q000172:deficiency / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D000939:Epitopes; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell; C496619:Vav1 protein, mouse