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.
Engagement of the T cell antigen receptor (TCR) by cognate peptide major histocompatibility complex (pMHC) triggers the assembly of the immunological synapse (IS), a dynamic supra-molecular membrane structure formed at the contact between the T cell and antigen-presenting cell (APC) (Kumari et al., 2014; Fooksman et al., 2010). In addition to organizing signal propagation, the IS likely participates in signal processing required for adequate T cell activation (Acuto et al., 2008). Over the last decade, intracellular membrane trafficking has been recognized as a key mechanism in the assembly and the function of IS, where it contributes to regulate signal intensity and duration by controlling the amount and dwell time of receptors and signalling mediators at the T cell–APC interface. The synaptic accumulation of endosomal vesicles containing TCRs, as well as other proteins that undergo cycles of internalization and re-expression, is achieved by polarized endosome recycling (Das et al., 2004; Soares et al., 2013a; Finetti and Baldari, 2013). Once delivered in the proximity of the IS environment, the TCRs continue to recycle between the plasma membrane and the recycling compartment. This ensures a continuous supply of endosome-associated receptors to the IS, until signalling is shut off as a result of their lysosomal degradation (Das et al., 2004). Other receptors, such as the transferrin receptor (TfR), CD28, LFA-1 (lymphocyte function-associated antigen 1, a complex between integrin αL and integrin β2), GLUT-1 (also known as SLC2A1) and CTLA-4 exploit the recycling pathway to move in or out of the IS (Yokosuka et al., 2008; Piotrowski et al., 2013; Finetti and Baldari, 2013; Egen and Ellison, 2002). Two key membrane-associated TCR signalling regulators, the kinase Lck and the transmembrane scaffold LAT, exist as two pools, one of which is associated with the plasma membrane and the other with recycling endosomes that translocate to the T-cell–APC interface in response to stimulation (Ley et al., 1994; Bonello et al., 2004; Ehrlich et al., 2002; Larghi et al., 2013; Soares et al., 2013b). Observations made by super-resolution microscopy show that, similar to the comparmentalization in distinct nanodomains of their surface counterparts (Lillemeier et al., 2010; Williamson et al., 2011; Sherman et al., 2011; Rossy et al., 2013), vesicular CD3ζ (also known as CD247), LAT and Lck are associated with distinct exocytic compartments marked by specific sets of Rab proteins, and are released at the IS in a differentially regulated manner (Soares et al., 2013b). It has been speculated that fusion at the IS of vesicles carrying these signalling molecules can generate nanoterritories that function as hubs for signal amplification (Soares et al., 2013b).
The diversification and complexity of the intracellular trafficking pathways converging to the IS poses a major challenge in dissecting the underlying mechanisms and identifying the dedicated proteins for each pathway. We have previously demonstrated that the IFT20, a component of intraflagellar transport (IFT) system, which is responsible for the assembly of the primary cilium in other cells, controls TCR accumulation at the IS in the non-ciliated T cell as a complex with IFT52, IFT57 and IFT88 (Finetti et al., 2009, 2014). This function involves the participation of IFT20 in the pathway that orchestrates polarized receptor recycling to the IS, with IFT20 interacting with the small GTPase Rab5 to promote the transit of internalized TCRs and TfRs from early to recycling endosomes (Finetti et al., 2009, 2014). Interestingly, we identified two other small GTPases, Rab8, a master regulator of ciliogenesis (Nachury et al., 2011), and Rab29, which is involved in Salmonella-containing vacuole trafficking in infected epithelial cells (Spanò et al., 2011), as central players in the TCR recycling pathway orchestrated by IFT20 and Rab11 (note that Rab5, Rab8 and Rab11 have more than one isoform, but we do not refer to a specific form here) (Finetti et al., 2015; Onnis et al., 2015). Of note, both Rab8 and Rab29 are dispensable for TfR recycling, while they are required for the recycling of CXCR4, which is not regulated by IFT20 (Finetti et al., 2015; Onnis et al., 2015). These data suggest the existence of multiple specialized pathways that intersect by combining shared regulators to control recycling of specific receptors in T cells. Data obtained in a CD4 T-cell-specific conditional IFT20−/− mouse have extended and validated in vivo the role of IFT20 in the assembly of a functional IS, implicating IFT20 also in the traffic of vesicular LAT (Vivar et al., 2016).
To further characterize the recycling pathway responsible for endosomal TCR trafficking to the IS, here, we used an unbiased approach to define novel IFT20 interactors by quantitative mass spectrometry (MS). We identified seven binding partners of IFT20, which included two interactors previously identified in T cells, i.e. IFT57 and IFT88 (Finetti et al., 2009, 2014), and five new interactors, namely 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). Of these, three were found to be required for TCR trafficking to the IS.
We undertook to identify IFT20-interacting partners by quantitative MS. A Tween StrepTagII was adjoined to the IFT20 C-terminus (IFT20–OST) to rapidly and quantitatively capture it and maximize recovery of protein partners. Total lysates of Jurkat T cells stably expressing IFT20–OST were subjected to pulldown using StrepTactin, eluted with biotin and analysed by nano-liquid chromatography tandem MS (nano-LC-MS/MS) (Fig.1A). StrepTactin pulldown in Jurkat cells not expressing IFT20–OST was used as a negative control. Data were analysed by label-free quantification using the MaxQuant software. Only proteins detected in none of three replicates in the negative control and that had a >2-fold abundance over control sample were considered as likely interactors.
