The T cell IFT20 interactome reveals new players in immune synapse assembly.
Sustained signalling at the immune synapse (IS) requires the synaptic delivery of recycling endosome-associated T cell antigen receptors (TCRs). IFT20, a component of the intraflagellar transport system, controls TCR recycling to the IS as a complex with IFT57 and IFT88. Here, we used quantitative mass spectrometry to identify additional interaction partners of IFT20 in Jurkat T cells. In addition to IFT57 and IFT88, the analysis revealed new binding partners, including IFT54 (also known as TRAF3IP1), GMAP-210 (also known as TRIP11), Arp2/3 complex subunit-3 (ARPC3), COP9 signalosome subunit-1 (CSN1, also known as GPS1) and ERGIC-53 (also known as LMAN1). A direct interaction between IFT20 and both IFT54 and GMAP-210 was confirmed in pulldown assays. Confocal imaging of antigen-specific conjugates using T cells depleted of these proteins by RNA interference showed that TCR accumulation and phosphotyrosine signalling at the IS were impaired in the absence of IFT54, ARPC3 or ERGIC-53. Similar to in IFT20-deficient T cells, this defect resulted from a reduced ability of endosomal TCRs to polarize to the IS despite a correct translocation of the centrosome towards the antigen-presenting cell contact. Our data underscore the traffic-related role of an IFT20 complex that includes components of the intracellular trafficking machinery in IS assembly.
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Donatella
D
Galgano
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Anna
A
Onnis
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Elisa
0000-0001-6881-9176
E
Pappalardo
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena 53100, Italy.
Federico
F
Galvagni
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
Oreste
O
Acuto
Department of Life Sciences, University of Siena, Siena 53100, Italy cosima.baldari@unisi.it.
Cosima T
0000-0002-4414-6744
CT
Baldari
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D054992:Immunological Synapses / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology; D037241:Mannose-Binding Lectins / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D022101:Microtubule-Organizing Center / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D060066:Protein Interaction Maps; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
C517043:ARPC3 protein, human; D051376:Actin-Related Protein 2-3 Complex; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C487991:IFT20 protein, human; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell; D011990:Receptors, Transferrin; C412724:TRAF3IP1 protein, human; C109331:TRIP11 protein, human
Immune synapse assembly; Intraflagellar transport system; Mass spectrometry analysis