Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.

• MeSH
• adaptorové proteiny signální transdukční imunologie   metabolismus   MeSH
• aktivace lymfocytů * MeSH
• fosfolipasa C gama metabolismus   MeSH
• fosforylace imunologie   MeSH
• ligandy MeSH
• membránové proteiny imunologie   metabolismus   MeSH
• myši MeSH
• protein-tyrosinkináza ZAP-70 metabolismus   MeSH
• receptory antigenů T-buněk imunologie   metabolismus   MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

• MeSH
• buněčné kultury MeSH
• cytokiny genetika   MeSH
• dánio pruhované krev   MeSH
• hematopoetické kmenové buňky cytologie   MeSH
• hematopoéza * MeSH
• kapři krev   MeSH
• kultivační média chemie   MeSH
• molekulární biologie metody   MeSH
• proteiny dánia pruhovaného genetika   MeSH
• rekombinantní proteiny genetika   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.

• MeSH
• buněčná diferenciace MeSH
• dánio pruhované embryologie   fyziologie   MeSH
• embryo nesavčí MeSH
• geneticky modifikovaná zvířata MeSH
• hematopoetické kmenové buňky fyziologie   MeSH
• hematopoéza genetika   MeSH
• kultivované buňky MeSH
• proliferace buněk MeSH
• thrombopoetin genetika   MeSH
• trombocyty fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• aplastická anemie MeSH
• kmenové buňky MeSH
• Publikační typ
• sborníky MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• biologie
• onkologie
• hematologie a transfuzní lékařství