real-time-live-imaging-of-t-cell-signaling-complex-formation

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Bar Ilan University

Bar-Ilan University

Protein-protein interactions regulate and control many cellular functions. A multimolecular complex consisting of the adaptor proteins SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kD), Nck, and the guanine nucleotide exchange factor Vav1 is recruited to the T cell side of the interface with an antigen-presenting cell during initial T cell activation. This complex is crucial for regulation of the actin machinery, antigen recognition, and signaling in T cells. We studied the interactions between these proteins as well as the dynamics of their recruitment into a complex that governs cytoskeletal reorganization. We developed a triple-color FÃ¶rster resonance energy transfer (3FRET) system to observe the dynamics of the formation of this trimolecular signaling complex in live human T cells and to follow the three molecular interactions in parallel. Using the 3FRET system, we demonstrated that dimers of Nck and Vav1 were constitutively formed independently of both T cell activation and the association between SLP-76 and Nck. After T cell receptor stimulation, SLP-76 was phosphorylated, which enabled the binding of Nck. A point mutation in the proline-rich site of Vav1, which abolishes its binding to Nck, impaired actin rearrangement, suggesting that Nck-Vav1 dimers play a critical role in regulation of the actin machinery. We suggest that these findings revise the accepted model of the formation of a complex of SLP-76, Nck, and Vav1 and demonstrate the use of 3FRET as a tool to study signal transduction in live cells.

Studying the dynamics of SLP-76, Nck, and Vav1 multimolecular complex formation in live human cells with triple-color FRET.

Interactions de prot&eacute;ine-prot&eacute;ine r&eacute;glementent et de contr&ocirc;lent de nombreuses fonctions cellulaires. Un complexe multimol&eacute;culaire compos&eacute; des prot&eacute;ines adaptateur SLP-76 (Src homologie 2 domaine leucocytes prot&eacute;ine contenant du 76 kD), Nck et le facteur d&#39;&eacute;change de nucl&eacute;otide guanine Vav1 est recrut&eacute; au c&ocirc;t&eacute; de l&#39;interface avec une cellule pr&eacute;sentatrices au cours des lymphocytes T initiales d&#39;activation des cellules T. Ce complexe est crucial pour le r&egrave;glement de l&#39;actine machines, reconnaissance de l&#39;antig&egrave;ne et la signalisation dans les cellules T. Nous avons &eacute;tudi&eacute; les interactions entre ces prot&eacute;ines ainsi que la dynamique de leur recrutement dans un complexe qui r&eacute;git la r&eacute;organisation du cytosquelette. Nous avons d&eacute;velopp&eacute; un triple-couleur F&ouml;rster resonance &eacute;nergie transfert (3FRET) syst&egrave;me d&#39;observer la dynamique de la formation de cette signalisation trimol&eacute;culaire complexes dans des cellules T humaines vivantes et de suivre les trois interactions mol&eacute;culaires en parall&egrave;le. Gr&acirc;ce au syst&egrave;me 3FRET, nous avons d&eacute;montr&eacute; que les dim&egrave;res de Nck et Vav1 sont constitutivement form&eacute;s ind&eacute;pendamment de l&#39;activation des cellules T et l&#39;association entre SLP-76 et Nck. Apr&egrave;s une stimulation des r&eacute;cepteurs des lymphocytes T, SLP-76 est phosphoryl&eacute;e, qui a permis la liaison de Nck. Une mutation ponctuelle dans le site riche en proline de Vav1, qui abolit sa liaison &agrave; Nck, impaired r&eacute;arrangement de l&#39;actine, ce qui sugg&egrave;re que les dim&egrave;res Nck-Vav1 jouent un r&ocirc;le critique dans la r&eacute;gulation de la machinerie de l&#39;actine. Nous sugg&eacute;rons que ces constatations r&eacute;viser le mod&egrave;le accept&eacute; de la formation d&#39;un complexe de SLP-76, Nck et Vav1 et d&eacute;montrent l&#39;utilisation de 3FRET comme un outil pour &eacute;tudier la transduction du signal dans des cellules vivantes.

&Eacute;tudier la dynamique de la formation de complexes multimol&eacute;culaire SLP-76, Nck et Vav1 dans des cellules vivantes humaines avec triple-couleur frette.

Interazioni proteina-proteina regolano e controllano molte funzioni cellulari. Un multimolecular complesso composto da proteine adattatore SLP-76 (Src homology 2 dominio contenenti leucociti proteina di 76 kD), Nck e il fattore di scambio di nucleotide della guanina Vav1 &egrave; reclutato al lato delle cellule T dell&#39;interfaccia con un&#39;antigene-presentare cellula durante iniziale attivazione delle cellule T. Questo complesso &egrave; cruciale per la regolazione dell&#39;actina macchinari, riconoscimento dell&#39;antigene e segnalazione in cellule T. Abbiamo studiato le interazioni tra queste proteine, nonch&eacute; le dinamiche della loro assunzione in un complesso che governa la riorganizzazione del citoscheletro. Abbiamo sviluppato un triple-colore F&ouml;rster risonanza energetica (3FRET) sistema di trasferimento a osservare le dinamiche di formazione di questa segnalazione trimolecolare complesso nelle cellule T umane dal vivo e a seguire le tre interazioni molecolari in parallelo. Utilizzando il sistema di 3FRET, abbiamo dimostrato che dimeri di Nck e Vav1 sono costitutivamente formati indipendentemente l&#39;associazione tra SLP-76 e Nck e attivazione delle cellule T. Dopo la stimolazione del recettore delle cellule T, &egrave; stato fosforilato SLP-76, che attivata l&#39;associazione di Nck. Una mutazione puntiforme nel sito di Vav1, che abolisce la sua associazione a Nck, ricco di prolina alterato riarrangiamento dell&#39;actina, suggerendo che Vav1 Nck dimeri svolgono un ruolo critico nella regolazione della macchina dell&#39;actina. Suggeriamo che queste scoperte rivedere il modello accettato la formazione di un complesso di SLP-76, Nck e Vav1 e dimostrano l&#39;uso di 3FRET come uno strumento per lo studio di trasduzione del segnale in cellule vive.

Studiando la dinamica della SLP-76, Nck e Vav1 multimolecular formazione dei complessi nelle cellule umane dal vivo con tripla-colore FRET.

Actin polymerization is a fundamental cellular process regulating immune cell functions and the immune response. The Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor, which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. WASp interacting protein (WIP) was first discovered as the binding partner of WASp, through the use of the yeast two hybrid system. WIP was later identified as a chaperone of WASp, necessary for its stability. Mutations occurring at the WASp homology 1 domain (WH1), which serves as the WIP binding site, were found to cause the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). WAS manifests as an immune deficiency characterized by eczema, thrombocytopenia, recurrent infections, and hematopoietic malignancies, demonstrating the importance of WIP for WASp complex formation and for a proper immune response. WIP deficiency was found to lead to different abnormalities in the activity of various lymphocytes, suggesting differential cell-dependent roles for WIP. Additionally, WIP deficiency causes cellular abnormalities not found in WASp-deficient cells, indicating that WIP fulfills roles beyond stabilizing WASp. Indeed, WIP was shown to interact with various binding partners, including the signaling proteins Nck, CrkL and cortactin. Recent studies have demonstrated that WIP also takes part in non immune cellular processes such as cancer invasion and metastasis, in addition to cell subversion by intracellular pathogens. Understanding of numerous functions of WIP can enhance our current understanding of activation and function of immune and other cell types.

WIP Remodeling Actin behind the Scenes: How WIP Reshapes Immune and Other Functions.

Polym&eacute;risation de l&#39;actine est un cellulaire fondamental du processus r&eacute;gissant les fonctions des cellules immunitaires et la r&eacute;ponse immunitaire. La prot&eacute;ine de Wiskott - Aldrich, syndrome (WASp) est une nucl&eacute;ation de l&#39;actine promotion de facteur, qui est exclusivement exprim&eacute;e dans les cellules h&eacute;matopo&iuml;&eacute;tiques, o&ugrave; il joue un r&ocirc;le de r&eacute;glementation dans la dynamique du cytosquelette. Prot&eacute;ine d&#39;interaction WASp (WIP) a &eacute;t&eacute; d&eacute;couvert comme le partenaire de liaison du WASp, au moyen du syst&egrave;me de deux hybrides de levure. Travaux en cours sera plus tard identifi&eacute; comme un chaperon de gu&ecirc;pe, n&eacute;cessaire &agrave; sa stabilit&eacute;. Mutations qui se produisent au niveau du domaine d&#39;homologie 1 gu&ecirc;pe (WH1), qui sert de l&#39;accepteur WIP, ont &eacute;t&eacute; trouv&eacute;es pour causer le syndrome de Wiskott Aldrich &ndash; (processus Windows WAS) et thrombop&eacute;nie li&eacute;e &agrave; le X (XLT). &Eacute;TAIT manifeste comme un d&eacute;ficit immunitaire caract&eacute;ris&eacute; par l&#39;ecz&eacute;ma, thrombocytop&eacute;nie, infections r&eacute;currentes et des malignit&eacute;s h&eacute;matopo&iuml;&eacute;tiques, d&eacute;montrant l&#39;importance des travaux en cours pour la formation de complexes de gu&ecirc;pe et une r&eacute;ponse immunitaire ad&eacute;quate. Carence en WIP s&#39;est av&eacute;r&eacute; conduisent &agrave; diff&eacute;rentes anomalies dans l&#39;activit&eacute; des lymphocytes diff&eacute;rents, sugg&eacute;rant la diff&eacute;rentielles d&eacute;pendant des lymphocytes r&ocirc;les pour WIP. En outre, une carence en WIP provoque des anomalies cellulaires non trouv&eacute;s dans les cellules d&eacute;ficientes en gu&ecirc;pe, indiquant que WIP remplit des r&ocirc;les au-del&agrave; de stabiliser la gu&ecirc;pe. En effet, WIP a &eacute;t&eacute; montr&eacute; pour interagir avec les diff&eacute;rents partenaires de liaison, y compris les prot&eacute;ines de signalisation Nck, CrkL et cortactin. Des &eacute;tudes r&eacute;centes ont d&eacute;montr&eacute; que les WIP prend &eacute;galement part &agrave; des processus cellulaires non immunitaires tels que l&#39;invasion du cancer et des m&eacute;tastases, en plus de la subversion de la cellule par des pathog&egrave;nes intracellulaires. Compr&eacute;hension des nombreuses fonctions du WIP peut am&eacute;liorer notre compr&eacute;hension actuelle de l&#39;activation et la fonction du syst&egrave;me immunitaire et d&#39;autres types de cellules.

WIP transformant l&#39;actine dans les coulisses : Comment WIP remod&egrave;le les fonctions immunitaires et autres.

Polimerizzazione dell&#39;actina &egrave; un cellulare fondamentale processo che regolano le funzioni delle cellule immunitarie e la risposta immunitaria. La proteina di Wiskott - Aldrich-Syndroms (WASp) &egrave; una nucleazione di actina promozione fattore, che si esprime esclusivamente in cellule ematopoietiche, dove svolge un ruolo normativo chiave nella dinamica del citoscheletro. Proteina d&#39;interazione WASp (WIP) fu scoperta come partner di associazione di WASp, attraverso il sistema di due ibridi di lievito. WIP pi&ugrave; tardi venne identificato come un chaperone di WASp, necessarie per la sua stabilit&agrave;. Le mutazioni che si verificano presso il dominio di omologia 1 WASp (WH1), che serve come il sito di legame di WIP, sono state trovate per causare la sindrome di Wiskott &ndash; Aldrich (WAS) e trombocitopenia legata al X (XLT). ERA si manifesta come un&#39;immunodeficienza caratterizzata da eczema, trombocitopenia, infezioni ricorrenti e neoplasie ematopoietiche, dimostrando l&#39;importanza di WIP per formazione complessa WASp e per una corretta risposta immunitaria. Deficit di WIP &egrave; stato trovato a condurre a diverse anomalie nell&#39;attivit&agrave; dei vari linfociti, suggerendo differenziali dipendente dalla cella ruoli per WIP. Inoltre, il deficit WIP provoca anomalie cellulari che non si trovano in celle di WASp-carenti, indicando che WIP soddisfa ruoli di l&agrave; di stabilizzazione WASp. Infatti, WIP &egrave; stato mostrato di interagire con i vari partner di associazione, comprese le proteine di segnalazione Nck, CrkL e cortactin. Studi recenti hanno dimostrato che WIP partecipa anche non immuni cellulari processi come l&#39;invasione del tumore e metastasi, oltre alla sovversione delle cellule da agenti patogeni intracellulari. Comprensione delle numerose funzioni del WIP pu&ograve; migliorare la nostra comprensione corrente di attivazione e funzione del sistema immunitario e altri tipi di cellule.

Article	Year
Studying the dynamics of SLP-76, Nck, and Vav1 multimolecular complex formation in live human cells with triple-color FRET. Science signaling\| PubMed ID: 22534133	2012
WIP Remodeling Actin behind the Scenes: How WIP Reshapes Immune and Other Functions. International journal of molecular sciences\| PubMed ID: 22837718	2012

Elad Noy

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