The T cell IFT20 interactome reveals new players in immune synapse assembly

Abstract

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.

Dissecting the Vesicular Trafficking Function of IFT Subunits

“…In addition to Rab proteins and three IFT subunits (IFT52, IFT57, and IFT88), some new players were identified in IFT20 interactomes, which included IFT54, GMAP210, subunit-3 of Arp2/3 complex (ARPC3), subunit-1 of COP9 signalosome (CSN1), and ERGIC-53. Moreover, loss of IFT54, ARPC3, or ERGIC-53 led to failure of endosomal TCR and TfR accumulation at the IS, which was in accordance with that observed in IFT20-deficient T cells; this greatly increased the complexity and diversity of the vesicular trafficking pathways where IFT20 participated (Galgano et al, 2017). Another axis of IFT20-IFT54-microtubule was found to be exploited to move the recycling endosomes to the IS in T cells (Bizet et al, 2015).…”

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP‐1–mediated endocytosis

“…IFTs are linked to endocytosis independent of cilia. In T cells that do not form a cilium, but rather form an immune synapse responsible for the recycling of endosome-associated T-cell antigen receptors, IFT88 has been associated with polarized receptor recycling alongside IFT20, IFT57, and IFT52 (57)(58)(59)(60). Ongoing studies are exploring the roles of other components of the ciliome in protease activity and endocytosis.…”

Transcellular communication at the immunological synapse: a vesicular traffic-mediated mutual exchange

“…The TCR is localized in Rab3d + and Rab8b + endosomes 23 and its traffic to the IS is regulated by Rab29 25 , Rab35 26 , and Rab8a 27 . Surprisingly, the components of the intraflagellar transport (IFT) system, which have a well-known function in ciliogenesis, have been reported as essential for the delivery of the TCR and LAT to the IS and T-cell activation, even though lymphocytes are devoid of a primary cilium 28 – 31 . The complexity of the mechanism underlying the endosomal trafficking to the IS is further increased by a variety of regulators of both the microtubule and the actin cytoskeleton as well as components of the machinery involved in vesicle fusion with the synaptic membrane 2 , 32 .…”