Galgano Donatella, Onnis Anna, Pappalardo Elisa, Galvagni Federico, Acuto Oreste, Baldari Cosima T
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
J Cell Sci. 2017 Mar 15;130(6):1110-1121. doi: 10.1242/jcs.200006. Epub 2017 Feb 2.
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.
免疫突触（IS）处的持续信号传导需要回收内体相关的T细胞抗原受体（TCR）向突触的递送。IFT20是鞭毛内运输系统的一个组成部分，它作为与IFT57和IFT88的复合物，控制TCR向IS的循环利用。在这里，我们使用定量质谱法来鉴定Jurkat T细胞中IFT20的其他相互作用伙伴。除了IFT57和IFT88，分析还揭示了新的结合伙伴，包括IFT54（也称为TRAF3IP1）、GMAP-210（也称为TRIP11）、Arp2/3复合物亚基3（ARPC3）、COP9信号体亚基1（CSN1，也称为GPS1）和ERGIC-53（也称为LMAN1）。在下拉实验中证实了IFT20与IFT54和GMAP-210之间的直接相互作用。使用通过RNA干扰耗尽这些蛋白质的T细胞对抗原特异性结合物进行共聚焦成像显示，在没有IFT54、ARPC3或ERGIC-53的情况下，IS处的TCR积累和磷酸酪氨酸信号传导受损。与IFT20缺陷的T细胞类似，尽管中心体正确地向抗原呈递细胞接触部位移位，但这种缺陷是由于内体TCR向IS极化的能力降低所致。我们的数据强调了一个IFT20复合物在IS组装中与细胞内运输机制成分相关的运输作用。
