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In JoVE (1)
Other Publications (5)
Articles by Maor H. Pauker in JoVE
Real-time Live Imaging of T-cell Signaling Complex Formation
Elad Noy*1, Maor H. Pauker*1, Mira Barda-Saad1
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University
We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.
Other articles by Maor H. Pauker on PubMed
The EMBO Journal. Jul, 2010 | Pubmed ID: 20562827
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.
Molecular and Cellular Biology. Jul, 2011 | Pubmed ID: 21536650
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.
Communicative & Integrative Biology. Mar, 2011 | Pubmed ID: 21655432
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.
Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-color FRET
Science Signaling. Apr, 2012 | Pubmed ID: 22534133
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.
Molecular and Cellular Biology. Aug, 2012 | Pubmed ID: 22665495
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.