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

T&#x7D30;&#x80DE;&#x6297;&#x539F;&#x53D7;&#x5BB9;&#x4F53;&#xFF08;TCR&#xFF09;&#x306E;&#x95A2;&#x4E0E;&#x306F;T&#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x591A;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x8907;&#x5408;&#x4F53;&#x306E;&#x5F62;&#x6210;&#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x3002; SLP-76&#x3068;&#x306E;&#x8907;&#x5408;&#x4F53;&#x306E;&#x751F;&#x6210;&#x306F;&#x3001;NCK&#x3068;VAV1&#x306F;&#x3001;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x6A5F;&#x69CB;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;SLP-76&#x3001;NCK&#x3068;VAV1&#x8907;&#x5408;&#x4F53;&#x306E;&#x7D44;&#x6210;&#x3001;&#x5316;&#x5B66;&#x91CF;&#x8AD6;&#x3068;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x7279;&#x7570;&#x6027;&#x3092;&#x5B9A;&#x7FA9;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x3053;&#x306E;&#x8907;&#x5408;&#x4F53;&#x306F;&#x3001;2 NCK&#x3068;2 VAV1&#x5206;&#x5B50;&#x3092;1 SLP-76&#x5206;&#x5B50;&#x3092;&#x542B;&#x3080;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002; NCK&#x3068;VAV1&#x306E;&#x9593;&#x306E;&#x76F4;&#x63A5;&#x7684;&#x306A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306F;&#x3001;NCK&#x306E;C&#x672B;&#x7AEF;SH3&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068;VAV1 N&#x672B;&#x7AEF;SH3&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068;&#x306E;&#x9593;&#x306E;&#x7D50;&#x5408;&#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x308B;&#x3002; VAV1&#x306E;&#x7834;&#x58CA;&#xFF1A;NCK&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x6709;&#x5BB3;&#x306A;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x305F;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x91CD;&#x5408;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x65B0;&#x898F;&#x306A;&#x77E5;&#x898B;&#x306F;&#x3001;T&#x7D30;&#x80DE;&#x6D3B;&#x6027;&#x5316;&#x5F8C;&#x306E;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x91CD;&#x5408;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306B;&#x65B0;&#x305F;&#x306A;&#x5149;&#x3092;&#x5F53;&#x3066;&#x308B;&#x3002;

SLP-76&#x30B3;&#x30F3;&#x30D7;&#x30EC;&#x30C3;&#x30AF;&#x30B9;&#x5354;&#x540C;&#x7D44;&#x5408;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306F;&#x3001;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x91CD;&#x5408;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;

R&#xE9;cepteur de lymphocytes T antig&#xE8;ne (TCR) d&#x26;#39;engagement induit la formation de complexes de signalisation &#xE0; plusieurs prot&#xE9;ines essentielles pour la r&#xE9;gulation des fonctions des lymphocytes T. G&#xE9;n&#xE9;ration d&#x26;#39;un complexe de SLP-76, NCK et VAV1 est crucial pour la r&#xE9;gulation de la machine actine. Nous d&#xE9;finissons la composition, la stoechiom&#xE9;trie et la sp&#xE9;cificit&#xE9; des interactions dans le SLP-76, Nck et complexe VAV1. Nos donn&#xE9;es r&#xE9;v&#xE8;lent que ce complexe peut contenir un SLP-76 mol&#xE9;cule, deux et deux Nck VAV1 mol&#xE9;cules. Un interaction directe entre Nck et VAV1 est induits par la liaison entre l&#x26;#39;extr&#xE9;mit&#xE9; C-terminale du domaine SH3 de Nck et le VAV1 N-terminale du domaine SH3. Perturbation de l&#x26;#39;VAV1: l&#x26;#39;interaction Nck pr&#xE9;judicierait polym&#xE9;risation de l&#x26;#39;actine. Ces nouveaux r&#xE9;sultats apportent un nouvel &#xE9;clairage sur le m&#xE9;canisme de polym&#xE9;risation de l&#x26;#39;actine, apr&#xE8;s activation des lymphocytes T.

Interactions coop&#xE9;ratives au Complexe SLP-76 sont critiques pour polym&#xE9;risation de l&#x26;#39;actine

T-Zell-Antigen-Rezeptor (TCR)-Engagement induziert Bildung von multi-protein Signalgebung komplexen unerl&auml;sslich f&uuml;r die Regulierung der T-Zell-Funktionen. Generierung eines Komplexes von SLP-76, Nck und VAV1 ist entscheidend f&uuml;r die Regulierung der Aktin-Maschinen. Wir definieren die Zusammensetzung, St&ouml;chiometrie und Spezifit&auml;t Interaktionen im SLP-76, Nck und VAV1 komplexe. Unsere Daten zeigen, dass die Anlage eines SLP-76-Molek&uuml;ls, zwei Nck und zwei VAV1 Molek&uuml;le enthalten kann. Eine direkte Interaktion zwischen Nck und VAV1 ist durch Bindung zwischen der C-terminalen SH3-Dom&auml;ne des Nck und der VAV1 N-terminalen SH3-Dom&auml;ne vermittelt. Unterbrechung der VAV1:Nck Interaktion betroffen landschaftsbild Aktin-Polymerisation. Diese neuartige Erkenntnisse werfen ein neues Licht auf den Mechanismus der Aktin-Polymerisation nach T-Zell-Aktivierung.

Kooperative Wechselwirkungen der SLP-76-komplex sind entscheidend f&uuml;r die Aktin-Polymerisation.

Impegno del ricevitore (TCR) di cellule T antigene induce la formazione di complessi di segnalazione multi-protein essenziali per regolare le funzioni delle cellule T. Generazione di un complesso di SLP-76, Nck e VAV1 &egrave; cruciale per la regolazione della macchina dell&#39;actina. Definiamo la composizione, la stechiometria e la specificit&agrave; delle interazioni nella SLP-76, Nck e VAV1 complesse. I nostri dati rivelano che questo complesso pu&ograve; contenere una molecola SLP-76, due Nck e due molecole di VAV1. Un&#39;interazione diretta tra Nck e VAV1 &egrave; mediata dal legame tra il dominio C-terminale SH3 di Nck e il dominio SH3 VAV1 N-terminale. Interruzione dell&#39;interazione VAV1:Nck colpite deleterio polimerizzazione dell&#39;actina. Questi nuovi risultati gettano nuova luce sul meccanismo di polimerizzazione dell&#39;actina dopo l&#39;attivazione delle cellule T.

Interazioni cooperative presso il complesso di SLP-76 sono critiche per la polimerizzazione dell&#39;actina.

T &#x7EC6;&#x80DE;&#x6297;&#x539F;&#x53D7;&#x4F53; (TCR) &#x53C2;&#x4E0E;&#x8BF1;&#x5BFC;&#x5F62;&#x6210;&#x7684; multi-protein &#x4FE1;&#x53F7;&#x914D;&#x5408;&#x7269;&#x8C03;&#x8282; T &#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x4E00;&#x79CD;&#x590D;&#x6742;&#x7684; SLP 76&#x3001; Nck &#x548C; VAV1 &#x4E00;&#x4EE3;&#x7684;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x673A;&#x68B0;&#x8C03;&#x8282;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x6211;&#x4EEC;&#x5728; SLP 76&#x3001; Nck &#x548C;&#x590D;&#x6742;&#x7684; VAV1 &#x4E2D;&#x5B9A;&#x4E49;&#x7EC4;&#x6210;&#x3001; &#x5316;&#x5B66;&#x8BA1;&#x91CF;&#x5B66;&#x548C;&#x7279;&#x5F02;&#x6027;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x663E;&#x793A;&#x6B64;&#x590D;&#x6742;&#x53EF;&#x4EE5;&#x5305;&#x542B;&#x4E00;&#x4E2A; SLP 76 &#x5206;&#x5B50;&#x3001; &#x4E24;&#x4E2A; Nck &#x548C; VAV1 &#x4E24;&#x4E2A;&#x5206;&#x5B50;&#x3002;Nck &#x548C; VAV1 &#x4E4B;&#x95F4;&#x7684;&#x76F4;&#x63A5;&#x4E92;&#x52A8;&#x88AB;&#x4ECB;&#x5BFC;&#x7684; Nck C &#x7EC8;&#x7AEF; SH3 &#x57DF;&#x548C; VAV1 N &#x7EC8;&#x7AEF; SH3 &#x57DF;&#x4E4B;&#x95F4;&#x7684;&#x7ED1;&#x5B9A;&#x3002;Deleteriously VAV1:Nck &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x4E2D;&#x65AD;&#x5F71;&#x54CD;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x7684;&#x805A;&#x5408;&#x3002;&#x8FD9;&#x4E9B;&#x65B0;&#x7684;&#x53D1;&#x73B0;&#x65B0;&#x63ED;&#x793A;&#x7684;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x805A;&#x5408;&#x540E;&#x6FC0;&#x6D3B; T &#x7EC6;&#x80DE;&#x7684;&#x673A;&#x5236;&#x3002;

&#x808C;&#x52A8;&#x86CB;&#x767D;&#x805A;&#x5408;&#x7684;&#x5173;&#x952E;&#x5728; SLP 76 &#x590D;&#x6742;&#x7684;&#x5408;&#x4F5C;&#x4E92;&#x52A8;&#x3002;

T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.

Cooperative interactions at the SLP-76 complex are critical for actin polymerization.

T &#xC138;&#xD3EC; &#xD56D; &#xC6D0; &#xC218;&#xC6A9; &#xCCB4; (TCR) &#xC57D;&#xD63C; T &#xC138;&#xD3EC; &#xAE30;&#xB2A5; &#xC870;&#xC808;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; multi-protein &#xC2E0;&#xD638; &#xB2E8;&#xC9C0;&#xC758; &#xD615;&#xC131;&#xC744; &#xC720;&#xB3C4; &#xD55C;&#xB2E4;. SLP-76, Nck vav1&#xC758; &#xBCF5;&#xC7A1; &#xD55C;&#xC758; &#xC0DD;&#xC131;&#xC740; &#xB9D0;&#xB77C; &#xAE30;&#xACC4; &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; SLP-76, Nck &#xBC0F; &#xBCF5;&#xC7A1; &#xD55C; VAV1 &#xC870;&#xC131;, stoichiometry &#xBC0F; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xD2B9;&#xC774;&#xC131;&#xC744; &#xC815;&#xC758;&#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xACF5;&#xAC1C;&#xC774; &#xB2E8;&#xC9C0; &#xD558;&#xB098;&#xC758; SLP 76 &#xBD84;&#xC790;, &#xB450; Nck&#xC640; &#xB450; &#xAC1C;&#xC758; VAV1 &#xBD84;&#xC790; &#xD3EC;&#xD568; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. Nck&#xC640; VAV1 &#xAC04;&#xC758; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xC0C1;&#xD638; &#xC791;&#xC6A9; Nck C &#xD130;&#xBBF8;&#xB110; SH3 &#xB3C4;&#xBA54;&#xC778;&#xACFC; VAV1 N &#xB9E8;&#xB05D; SH3 &#xB3C4;&#xBA54;&#xC778; &#xAC04;&#xC758; &#xBC14;&#xC778;&#xB529; &#xC911;&#xC7AC;&#xC785;&#xB2C8;&#xB2E4;. VAV1:Nck &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xC7A5;&#xC560; deleteriously &#xB9D0;&#xB77C; &#xC911; &#xD569;&#xC5D0; &#xC601;&#xD5A5;&#xC744;. &#xC774; &#xC0C8;&#xB85C;&#xC6B4; &#xBC1C;&#xACAC;&#xC740; T &#xC138;&#xD3EC; &#xD65C;&#xC131;&#xD654; &#xD6C4; &#xB9D0;&#xB77C; &#xC911; &#xD569; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xBE5B;&#xC744; &#xB098;&#xD0C0;&#xB0C8;&#xB2E4;.

SLP 76 &#xBCF5;&#xC7A1; &#xD55C;&#xC5D0; &#xD611;&#xB825; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC740; &#xB9D0;&#xB77C; &#xC911; &#xD569;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

Receptor de c&#xE9;lulas T ant&#xED;geno (TCR) compromiso induce la formaci&#xF3;n de complejos de se&#xF1;alizaci&#xF3;n de m&#xFA;ltiples prote&#xED;nas esenciales para la regulaci&#xF3;n de las funciones de las c&#xE9;lulas T. Generaci&#xF3;n de un complejo de SLP-76, NCK y Vav1 es crucial para la regulaci&#xF3;n de la maquinaria de la actina. Se define la composici&#xF3;n, estequiometr&#xED;a y la especificidad de las interacciones en la SLP-76, NCK y compleja Vav1. Nuestros datos revelan que este complejo puede contener una mol&#xE9;cula de SLP-76, Nck dos y dos mol&#xE9;culas Vav1. Una interacci&#xF3;n directa entre Nck y Vav1 est&#xE1; mediada por la uni&#xF3;n entre el C-terminal de dominio SH3 de NCK y el Vav1 dominio N-terminal SH3. La interrupci&#xF3;n de la Vav1: la interacci&#xF3;n Nck perjudicialmente afectados polimerizaci&#xF3;n de la actina. Estos nuevos hallazgos arrojan nueva luz sobre el mecanismo de la polimerizaci&#xF3;n de la actina despu&#xE9;s de la activaci&#xF3;n de c&#xE9;lulas T.

Las interacciones cooperativas en el Complejo SLP-76 son esenciales para la polimerizaci&#xF3;n de actina

T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion- and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.

Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.

Activation des cellules t antig&egrave;ne r&eacute;cepteur (TCR) d&eacute;clenche des changements profonds dans le cytosquelette d&#39;actine. Outre le contr&ocirc;le de polarit&eacute; et la forme cellulaire, ce processus r&eacute;gule vital lymphocytes T, telles que l&#39;adh&eacute;rence des cellules T, la motilit&eacute; et la prolif&eacute;ration. Ceux-ci d&eacute;pendent du recrutement des prot&eacute;ines signalisation Nck et la maladie de Wiskott - Aldrich, syndrome prot&eacute;ine (WASp) sur le site d&#39;activation de TCR et sur les propri&eacute;t&eacute;s fonctionnelles de l&#39;&eacute;diteur de liens de prot&eacute;ines adaptateur pour l&#39;activation des cellules T (LAT) et SH2-domaine contenant des prot&eacute;ines de leucocytes de 76 kDa (SLP76). Maintenant, nous d&eacute;montrons que Nck est n&eacute;cessaire mais non suffisante pour le recrutement de gu&ecirc;pe. Nous montrons que les deux voies conduisent &agrave; r&eacute;arrangement d&#39;actine SLP76-d&eacute;pendante. On exige le domaine acide SLP76, crucial pour la liaison avec le domaine Nck SH2, et l&#39;autre le domaine SH2 SLP76, essentiel pour l&#39;interaction avec la prot&eacute;ine d&#39;adapteur adh&eacute;rence et d&eacute;granulation-promotion ADAP. Coop&eacute;ration fonctionnelle entre Nck et ADAP intervient dans les interactions SLP76-WASp et le r&eacute;arrangement de l&#39;actine. Aussi, nous r&eacute;v&eacute;lons le m&eacute;canisme mol&eacute;culaire qui relie ADAP &agrave; la r&eacute;organisation de l&#39;actine.

Coop&eacute;ration fonctionnelle entre les prot&eacute;ines Nck et ADAP est fondamentale pour la r&eacute;organisation de l&#39;actine.

Attivazione di cellule t antigene recettore (TCR) innesca cambiamenti profondi del citoscheletro di actina. Oltre a controllare la polarit&agrave; e la forma cellulare, questo processo regola risposte vitali delle cellule T, come ad esempio l&#39;adesione delle cellule T, la motilit&agrave; e la proliferazione. Queste dipendono dall&#39;assunzione di proteine segnalazione Nck e proteina Wiskott - Aldrich-Syndroms (WASp) al sito di attivazione TCR e sulle propriet&agrave; funzionali del linker proteine adattatore per l&#39;attivazione delle cellule T (LAT) e SH2-dominio-contenente proteine leucocitarie di 76 kDa (SLP76). Dimostriamo ora che Nck &egrave; necessaria, ma insufficiente per il reclutamento di WASp. Mostriamo che due percorsi conducono a riarrangiamento dell&#39;actina SLP76-dipendente. Uno richiede il dominio SLP76 acidulo, cruciale all&#39;associazione con il dominio Nck SH2, e un altro il dominio SH2 SLP76, essenziale per l&#39;interazione con la proteina di adesione e degranulazione promuovendo adattatore ADAP. Cooperazione funzionale tra Nck e ADAP media le interazioni SLP76-WASp e riarrangiamento dell&#39;actina. Vi sveliamo anche il meccanismo molecolare che collega ADAP alla riorganizzazione dell&#39;actina.

Cooperazione funzionale tra le proteine Nck e ADAP &egrave; fondamentale per la riorganizzazione dell&#39;actina.

Following T-cell antigen receptor (TCR) engagement, a multi-molecular complex consisting of SLP-76, Nck and VAV1 is formed and recruited to the T-cell antigen-presenting-cell (APC) interaction site. This complex is crucial for the regulation of the actin machinery. The molecules Nck (an adaptor) and VAV1 (a GEF for small G-proteins) were previously shown to bind SLP-76. Using high-resolution imaging techniques, together with gene silencing and biochemical analysis, we studied the dynamics of this signaling complex formation. We recently showed that VAV1 and Nck can bind each other independently of SLP-76. This direct interaction is mediated by the binding of the Nck C-terminal SH3 domain and the VAV1 N-terminal SH3 domain. This interaction contributes to the cooperative nature of the complex formation. This observation was confirmed in functional studies: disruption of the Nck-VAV1 interaction strongly inhibited actin polymerization. Here, we show that Nck-VAV1 interaction is not required for Ca(2+) mobilization, since a point mutation in the VAV1 N-terminal SH3 domain, which prevents the direct interaction between Nck and VAV1, has no effect on Ca(2+) flux and minimal effects on ZAP-70, LAT or PLCÎ³1 phosphorylation.

Studies of novel interactions between Nck and VAV SH3 domains.

Suite d&#39;engagement de r&eacute;cepteur (TCR) antig&egrave;ne de T-cellule, un multi-molecular complexe compos&eacute; de SLP-76, Nck et VAV1 est forme et recrute sur le site d&#39;interaction cellules T antig&egrave;ne-pr&eacute;sentation-cellule (APC). Ce complexe est crucial pour le r&egrave;glement de la machinerie de l&#39;actine. Les mol&eacute;cules Nck (un adaptateur) et VAV1 (un FEM pour petites prot&eacute;ines G) ont &eacute;t&eacute; pr&eacute;c&eacute;demment montr&eacute; pour lier SLP-76. En utilisant des techniques d&#39;imagerie &agrave; haute r&eacute;solution, ainsi que de silen&ccedil;age g&eacute;nique et des analyses biochimiques, nous avons &eacute;tudi&eacute; la dynamique de cette signalisation complexant. Nous avons montr&eacute; r&eacute;cemment que VAV1 et Nck peuvent lier l&#39;autre ind&eacute;pendamment de SLP-76. Cette interaction directe est m&eacute;di&eacute;e par la liaison du domaine C-terminal de Nck SH3 et le domaine SH3 VAV1 N-terminale. Cette interaction contribue &agrave; la nature coop&eacute;rative de la formation du complexe. Cette observation a &eacute;t&eacute; confirm&eacute;e dans des &eacute;tudes fonctionnelles : perturbation de l&#39;interaction de Nck-VAV1 inhib&eacute;e fortement la polym&eacute;risation de l&#39;actine. Ici, nous montrons que Nck-VAV1 interaction n&#39;est pas requise pour la mobilisation de Ca(2+), &eacute;tant une mutation ponctuelle dans le domaine SH3 VAV1 N-terminal, qui emp&ecirc;che l&#39;interaction directe entre Nck et VAV1, n&#39;a aucun effet sur le flux de Ca(2+) et des effets minimes sur la phosphorylation ZAP-70, LAT ou PLC&gamma;1.

&Eacute;tudes de nouvelles interactions entre domaines Nck et SH3 VAV.

Dopo il fidanzamento di cellule T antigene receptor (TCR), un multi-molecular complesso composto da SLP-76, Nck e VAV1 &egrave; formata e reclutato per il sito di interazione delle cellule T antigene-presentare-cella (APC). Questo complesso &egrave; cruciale per la regolazione della macchina dell&#39;actina. Le molecole Nck (un adattatore) e VAV1 (un GEF per piccole proteine G) erano precedentemente indicato associare SLP-76. Utilizzando tecniche di imaging ad alta risoluzione, insieme al silenziamento genico e analisi biochimica, abbiamo studiato la dinamica di questa segnalazione formazione complessa. Abbiamo recentemente dimostrato che VAV1 e Nck pu&ograve; associare l&#39;altro indipendentemente da SLP-76. Questa interazione diretta &egrave; mediata dal legame del dominio SH3 Nck C-terminale e il dominio SH3 VAV1 N-terminale. Questa interazione contribuisce alla natura cooperativa della formazione del complessa. Questa osservazione &egrave; stata confermata in studi funzionali: interruzione dell&#39;interazione Nck-VAV1 fortemente inibito la polimerizzazione dell&#39;actina. Mostriamo qui, che VAV1 Nck interazione non &egrave; richiesto per Ca(2+) mobilitazione, dal momento che una mutazione puntiforme del dominio SH3 VAV1 N-terminale, che impedisce l&#39;interazione diretta tra Nck e VAV1, ha alcun effetto sul flusso di Ca(2+) ed effetti minimi sulla fosforilazione di ZAP-70, LAT o PLC&gamma;1.

Studi del romanzo interazioni tra domini Nck e SH3 VAV.

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.

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.

Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics.

La prot&eacute;ine de Wiskott - Aldrich, syndrome (WASp) est un important r&eacute;gulateur de la dynamique de l&#39;actine durant l&#39;adh&eacute;rence et la motilit&eacute; cellulaire, et les mutations dans le g&egrave;ne sont responsables pour syndrome de Wiskott - Aldrich (WAS). Ici, nous d&eacute;montrons que la gu&ecirc;pe est ubiquitylated suivant l&#39;activation des lymphocytes T antig&egrave;ne r&eacute;cepteur (TCR). Phosphorylation de gu&ecirc;pe &agrave; la tyrosine 291 r&eacute;sultats en mati&egrave;re de recrutement de la ligase E3 Cbl-b, qui, ainsi que c-Cbl, effectue des WASp ubiquitination. R&eacute;sidus de lysine, 76 et 81, situ&eacute; au niveau du domaine de gu&ecirc;pe WH1, qui contient la grande majorit&eacute; des mutations du g&egrave;ne WASp, servent les sites de l&#39;ubiquitination. Perturbation de la gu&ecirc;pe non-d&eacute;gradante provoque l&#39;accumulation de la gu&ecirc;pe et modifie la dynamique de l&#39;actine et la formation des structures de l&#39;actine-d&eacute;pendante. Nos r&eacute;sultats sugg&egrave;rent que la d&eacute;gradation r&eacute;glement&eacute;e de gu&ecirc;pe activ&eacute; pourrait &ecirc;tre une strat&eacute;gie efficace par lequel la dur&eacute;e et la localisation de r&eacute;arrangement de l&#39;actine et l&#39;intensit&eacute; de l&#39;activation des lymphocytes T sont contr&ocirc;l&eacute;s.

R&eacute;gulation n&eacute;gative non-d&eacute;gradante d&eacute;pendant de gu&ecirc;pe est essentielle pour la dynamique de cytosquelette d&#39;actine.

La proteina di Wiskott - Aldrich-Syndroms (WASp) &egrave; un regolatore chiave della dinamica di actina durante l&#39;adesione e la motilit&agrave; cellulare, e mutazioni nel suo gene sono responsabili della sindrome di Wiskott &ndash; Aldrich (WAS). Qui, ci dimostrano che la Vespa &egrave; ubiquitylated dopo l&#39;attivazione di cellule T antigene recettore (TCR). La fosforilazione di Vespa a tirosina 291 risultati nel reclutamento della ligasi E3 Cbl-b, che, insieme a c-Cbl, effettua ubiquitylation WASp. Residui di lisina 76 e 81, situato presso il dominio WASp WH1, che contiene la maggior parte delle mutazioni del gene WASp, servono come i siti di ubiquitylation. Interruzione del WASp ubiquitylation provoca accumulo di WASp e altera la dinamica dell&#39;actina e la formazione di strutture di actina-dipendente. I nostri dati suggeriscono che regolamentato degradazione di WASp attivata potrebbe essere una strategia efficace da cui sono controllate la durata e l&#39;intensit&agrave; dell&#39;attivazione delle cellule T e localizzazione di riarrangiamento dell&#39;actina.

Article	Year
Cooperative interactions at the SLP-76 complex are critical for actin polymerization. The EMBO journal\| PubMed ID: 20562827	2010
Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization. Molecular and cellular biology\| PubMed ID: 21536650	2011
Studies of novel interactions between Nck and VAV SH3 domains. Communicative & integrative biology\| PubMed ID: 21655432	2011
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
Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics. Molecular and cellular biology\| PubMed ID: 22665495	2012

Maor H. Pauker

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