Surface expression of plasma membrane (PM)-associated receptors is dynamically regulated through constitutive or ligand-dependent endocytosis. Receptor internalization, which occurs in a clathrin-dependent or clathrin-independent manner (Doherty and McMahon, 2009), results in their targeting to the endocytic pathway. This pathway is orchestrated by a series of intracellular membrane-bound compartments that allow for the sorting of these molecules, referred to as cargoes, for one of two alternative fates: delivery to lysosomes or vacuoles for degradation by the endosomal sorting complex required for transport (ESCRT) and the multivesicular bodies (MVBs) compartment (Vietri et al., 2020) or targeting to the trans-Golgi network (TGN) or to the PM for reuse (Johannes and Wunder, 2011; Hsu et al., 2012). In this second route, the cargo is first recognized by a retrieval complex and routed away from the degradative pathway, then is pinched off from the endosome as a vesicle and coupled to cytoskeletal motor proteins for delivery to the target compartment (Burd and Cullen, 2014; Wang et al., 2018).

Sorting of endosomal cargo for recycling relies on a number of multiprotein complexes spatially and temporally regulated. The two main complexes responsible for endosomal retrieval are the retromer complex (Burd and Cullen, 2014) and the more recently identified retriever complex acting together with the CCC complex (Phillips-Krawczak et al., 2015; McNally et al., 2017). The Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex plays essential roles in both retromer- and retriever-dependent pathways by promoting branched actin polymerization on endosomes (Gomez and Billadeau, 2009; Phillips-Krawczak et al., 2015; McNally et al., 2017). These complexes are not only important for the trafficking of molecules from the PM to the compartments of destination but also play a key role in polarized recycling of specific molecules to specialized areas of the cell as observed, for example, in T lymphocytes undergoing immune synapse (IS) formation. Indeed, upon T-cell receptor (TCR) engagement, endosomal trafficking is redirected toward the contact area of the T cell with the antigen-presenting cell (APC) by local F-actin pools that act in concert with microtubules and endosomal traffic regulators (Soares et al., 2013; Martín-Cófreces and Sánchez-Madrid, 2018; Onnis and Baldari, 2019). Here, we will review the role of the main molecular complexes involved in endosomal cargo recycling and the related actin dynamics, with a focus on polarized recycling to the T-cell IS.

Endosomes are cellular hubs where internalized cargoes are sorted toward different trafficking pathways. Some cargoes are routed to the PM by recycling endosomes, a process known as endosome-to-plasma membrane recycling; others are transported to the TGN through an endosome-to-TGN retrieval or retrograde transport. Cargo recycling back to the cell surface can occur either via a fast recycling pathway controlled by the small GTPase Rab4 or via a slow recycling pathway in a Rab11-dependent manner (Galvez et al., 2012; Wandinger-Ness and Zerial, 2014; Figure 1A).

Endosomal sorting is accompanied by endosomal maturation. Early endosomes (EEs) are the main sorting station in the cell. EEs are characterized by specific markers such as Rab5 and early endosome antigen 1 (EEA1) and by the presence of large domains enriched in phosphatidylinositol(3,4,5)-triphosphate (PIP3) and sortin nexin (SNX) family members. A remarkable mosaicism in the EE membrane has emerged with the finding that cargoes, once they have reached the EEs, are targeted for degradation or recycling through the formation of specialized membrane subdomains that allow for cargo sorting and routing to the respective trafficking pathways through the local recruitment of specific molecular assemblies (Sönnichsen et al., 2000; Puthenveedu et al., 2010). The molecular machinery essential for cargo sorting in the recycling pathway is represented by three main complexes: retromer, retriever, and the CCC complex.

The WASH complex is a pentameric complex composed of WASH1 (WASHC1), Strumpellin (WASHC5), the Strumpellin and WASH-interacting protein SWIP (also known as KIAA1033 or WASHC4), FAM21A/C (family with sequence similarity 21A and C, also known as WASHC2A/C), and coiled-coil domain containing protein 53 (CCDC53 or WASHC3) (Derivery et al., 2009; Gomez and Billadeau, 2009; Jia et al., 2010; Alekhina et al., 2017; Figure 1C). Among these, FAM21 is an important structural component of the WASH complex for its key role in interacting with other protein complexes through its long, unstructured C-terminal tail containing multiple functional binding sites consisting of 21 copies of the LFa motif, rich in leucine, phenylalanine, and several acidic residues (Derivery and Gautreau, 2010). FAM21 associates with multiple VPS35 retromer subunits (Harbour et al., 2012; Jia et al., 2012; Helfer et al., 2013), as well as with the CCDC93 subunit of the CCC complex (Phillips-Krawczak et al., 2015), thereby coupling both retromer and retriever to endosomes. FAM21 also contains two regions within its tail that are able to bind with intermediate affinity to PI3P and with strong affinity to PI(3,5)P2 (Singla et al., 2019). Moreover, FAM21 can associate with the CAPZα/β heterodimer, known as capping protein (CP). CP binds to the barbed end of the actin protofilament, thereby controlling filament growth by inhibiting monomer addition or loss from that end. FAM21 interacts directly with CAPZ and impairs its actin-capping activity (Hernandez-Valladares et al., 2010).

WASH1 is another important component of the WASH complex that serves as nucleation-promoting factor (NPF) by activating Arp2/3-dependent actin polymerization on endosomal membranes (Derivery et al., 2009; Gomez and Billadeau, 2009; Jia et al., 2010). Arp2/3 is a heptameric protein complex, so called because of its two main components, actin-related proteins (Arp) 2 and 3. It is the first actin nucleator identified in eukaryotic cells and is highly conserved among species (Pizarro-Cerdá et al., 2017). The ability of WASH to activate the Arp2/3 complex is finely tuned by ubiquitination (Hao et al., 2013). The E3 ubiquitin ligase TRIM27 and its enhancer MAGE-L2 are recruited by the retromer subunit VPS35 to WASH1, resulting in its K63-linked polyubiquitination, which leads to a conformational change that enhances actin nucleation (Hao et al., 2013). WASH ubiquitination is further regulated by the USP7 enzyme, which has a dual activity: to promote WASH ubiquitination by preventing TRIM27 auto-ubiquitination and degradation and, concomitantly, to limit WASH ubiquitination through its direct deubiquitination (Hao et al., 2015).

Another activator of the Arp2/3 complex that has been described to associate with endosomes is cortactin (Kaksonen et al., 2000; Lladó et al., 2008). Cortactin controls a wide range of processes including the maturation of late endosomes and lysosomes, the retrograde transport to the Golgi apparatus, and actin dynamics at endosomes. Cortactin is a class II NPF that promotes actin assembly both by inducing Arp2/3-dependent actin polymerization and by binding and stabilizing pre-existing branched F-actin nucleated by the WASH complex. Cortactin is in turn regulated by PI(3,5)P2, which directly interacts with its actin-binding domain, preventing F-actin binding and leading to the inhibition of cortactin-mediated branched F-actin nucleation and stabilization (Hong et al., 2015). Interestingly, the ability of PIs to regulate F-actin dynamics is not limited to PI(3,5)P2. For instance, PI(4,5)P2, the best characterized actin regulator, interacts with and modulates N-WASP and actin-binding proteins such as cofilin, CAPZ, filamin, vinculin, talin, and others (Yin and Janmey, 2003). On the other hand, PI(3,4,5)P3 regulates the activation of the WASP family member WAVE2 to control lamellipodial protrusion (Suetsugu et al., 2006), highlighting a pleiotropic role of PIs in actin cytoskeleton regulation (Saarikangas et al., 2010).

Endosome sorting and maturation is accompanied by continuous membrane remodeling that requires the participation of both the actin and microtubule cytoskeletons. F-Actin is implicated in this process starting from the earliest steps, participating in defining endosomal subdomains to establish cargo destination. F-Actin associates with recycling microdomains and prevents the loss of recycling cargo to the degradative machinery (Simonetti and Cullen, 2019). In addition, F-actin polymerization at endosomes is essential for cargo sorting to recycling endosomes. Until recently, cargo recycling was believed to occur through sequence-independent “bulk” flow, as in the case of the transferrin receptor (Maxfield and McGraw, 2004). However, a large variety of cargoes have been demonstrated to recycle through a sequence-dependent pathway tightly regulated by actin dynamics (Puthenveedu et al., 2010; Temkin et al., 2011; Burd and Cullen, 2014). Cargoes to be recycled through this pathway are first recognized by a retrieval complex (retromer or retriever) and then packaged into tubulo-vesicular transport carriers enriched in F-actin and actin-related proteins (Puthenveedu et al., 2010). At this stage, microtubules join the game, with the microtubule-associated motor dynein and its partner dynactin, allowing for the extension of the nascent carriers. The WASH complex participates also in this step, exploiting its ability to interact with tubulin to stabilize the carriers on microtubule tracks. Subsequently, WASH-dependent F-actin nucleation at the apical portion of the tubular carriers provides the pushing force necessary for membrane fission. Following their detachment, F-actin regulates the short-distance mobility of tubular carriers mainly through actin motors such as myosin (Derivery et al., 2009; Harbour et al., 2010; Figure 2). This is exemplified by the β2AR receptor, which is recycled via tubular profiles enriched in F-actin and actin-related machineries (Bowman et al., 2016). A key role in this process is played by cortactin, which participates in the signaling cascade that regulates recycling (Vistein and Puthenveedu, 2014), the actin binding protein filamin A (FLNa) responsible for cargo entry into the tubular recycling domains (Pons et al., 2017), and other actin regulators including formins (Gong et al., 2018).

Following sorting at EEs, other recycling cargoes, such as the CI-MPR or sortilin, undergo retrograde trafficking to the TGN (Tu et al., 2020). In this process, cargoes are recognized by the SNX3–retromer complex, SNX-BARs, or clathrin and the adapter protein AP1, confined to specific endosomal subdomains and, following endosomal fission, transported by tubulo-vesicular transport carriers toward the TGN along microtubule tracks (Lu and Hong, 2014; Cheung and Pfeffer, 2016; Saimani and Kim, 2017). Once they arrive close to the TGN compartment, cargoes are captured by TGN-localized golgin proteins, such as golgin-97, golgin-245, GCC88, and GCC185 (Lowe, 2019). This process also involves TBC1D23, a protein that acts as a bridging protein by binding simultaneously to golgins and to the WASH complex subunit FAM21 on endosomal vesicles (Shin et al., 2017). Finally, carrier fusion with the Golgi membrane is mediated by four different vSNARE–tSNARE complexes (Lu and Hong, 2014).

Membrane trafficking is orchestrated by a variety of Rab GTPases that control different steps of the process, from cargo sorting to vesicle budding, motility, and fusion, through the recruitment of effector molecules, including the actin regulators mentioned in the previous paragraphs (Stenmark, 2009). Rab proteins are important regulators of retromer-mediated vesicular transport, not only in mammals but also in other organisms. For example, the core complex of retromer was found to interact with the GTP-bound form of Rab7 (Rab7-GTP) in yeast, plants, and mammalian cells (Rojas et al., 2008; Liu et al., 2012; Zelazny et al., 2013), leading to retromer recruitment to late endosomes (Priya et al., 2015). Rab32 regulates the retrograde trafficking of the CI-MPR to the TGN by directly interacting with SNX6 (Waschbüsch et al., 2019), while Rab21 is implicated in cargo sorting by establishing a complex with WASH and retromer to regulate endosomal F-actin (Del Olmo et al., 2019). Additionally, Rab9, together with retromer, WASH, and F-actin, has been recently reported to form an endosomal retrieval machinery that regulates selective recycling of the luminal protein Serpentine in the Drosophila trachea (Dong et al., 2013). Although little is known about the interplay between Rab GTPases and the retriever and CCC and WASH complexes, a recent proteomic study focused on SNARE and Rab proteins identified functional clusters, such as a correlation between Rab10 and the SNARE Syntaxin4 (STX4) or between Rab7/Rab21 and the WASH and CCC complexes (Clague and Urbé, 2020).

Rab GTPases also participate in tethering of the vesicles carrying recycling cargo to the target membrane through the recruitment of tethering factors (Stenmark, 2009). Rab32 and Rab38 were found to be implicated in the trafficking of the glucose transporter GLUT1 to the PM by regulating the effector molecule VARP, which in turn binds to the R-SNARE VAMP7 to facilitate membrane fusion between recycling endosomes carrying GLUT1 and the PM (Hesketh et al., 2014). SNX1 has been reported to interact with Rab6IP, a Rab6-interacting protein localized at the Golgi compartment and involved in the tethering of endosome-derived transport carriers to the TGN (Miserey-Lenkei et al., 2007). A similar function was observed for the Drosophila orthologue of TBC1D23, tbc1, a Rab GTPase-activating protein that couples endosome-derived vesicles to their target membrane at the TGN (Johnson and Andrew, 2019).

Although the specific identities and roles of Rab GTPases during receptor recycling are only beginning to be investigated, these findings underscore a tight functional interplay of these membrane trafficking regulators with the sorting, transport, and tethering machineries at all critical steps in receptor recycling. While the link between these Rabs and the cargo sorting complexes supports their implication in the local regulation of F-actin dynamics, the underlying mechanisms remain to be directly addressed.

The molecular complexes described above and the associated actin dynamics participate in cell polarization by regulating the recycling-dependent accumulation of receptors, adhesion molecules, and signaling mediators at specialized areas of the PM. This is exemplified by the formation of apical membrane specializations including primary cilia and apical microvilli of ciliated cells (Goldenring, 2015), the polarization of epithelial or neuronal cells (Vergés, 2016), or immune synapse (IS) formation in T lymphocytes and other immune cells.

The IS can be described as a highly polarized structure that forms at the T-cell interface with an APC carrying cognate MHC-bound antigen and allows the communication between the two cells to ensure efficient TCR signal transduction and T-cell activation (Dustin and Choudhuri, 2016). The typical “bull’s eye” structure of the mature IS features three concentric regions, referred to as supramolecular activation clusters (SMACs) that can be distinguished based on the specific partitioning of TCRs, costimulatory molecules, and integrins: the TCR-enriched central SMAC (cSMAC), the integrin-enriched peripheral SMAC (pSMAC), and the distal SMAC (dSMAC), where molecules with large ectodomains and negative regulators of TCR signaling are confined. IS assembly is coordinated by both the actin and the microtubule cytoskeletons, which drive the accumulation and partitioning of the synaptic components throughout the extended timeframe required for T-cell activation (Ritter et al., 2013; Martín-Cófreces and Sánchez-Madrid, 2018; Hammer et al., 2019).

The actin cytoskeleton plays a key role beginning from the first step of IS formation, which involves the assembly of TCR microclusters that move centripetally from the periphery to the center of the IS using F-actin as driving force (Ritter et al., 2013). In addition, actin promotes the activation of integrins to stabilize the T cell–APC contact and forms a ring-like seal at the inner side of the dSMAC (Hammer et al., 2019). However, the role of F-actin in IS assembly and function extends beyond the rearrangement and signaling events that occur at the PM. Early upon TCR activation, the centrosome translocates toward the IS in a process that is in part regulated by centrosomal F-actin dynamics (Ritter et al., 2013). Centrosome polarization is coordinated with F-actin clearance to generate a central F-actin-free area that facilitates the polarized release of effector molecules, such as cytokines produced by helper or cytotoxic T cells or the cytotoxic contents of the lytic granules of CTLs (Hivroz et al., 2012; Ritter et al., 2015; de la Roche et al., 2016; Kabanova et al., 2018; Herranz et al., 2019; Sanchez et al., 2019). Synaptic F-actin dynamics is also essential for the polarized release of vesicles carrying bioactive molecules, including synaptic ectosomes, which are assembled and released directly from the PM (Saliba et al., 2019), and exosomes, which are released upon the fusion of MVBs with the PM (Mittelbrunn et al., 2015; Bello-Gamboa et al., 2020).

Following polarization, the centrosome rapidly generates a network of microtubules both irradiating from the centrosome toward the periphery of the IS and converging from the periphery toward the center of IS to guide the polarized transport of vesicular components and organelles (Martín-Cófreces and Sánchez-Madrid, 2018). The identity of the vesicle-associated molecules that undergo polarized exocytosis to the IS depends on the type of T lymphocyte and APC, with helper T cells (CD4⁺ cells) secreting cytokines at the IS formed with cognate MHC-II-bearing cell targets to promote their maturation and function and cytotoxic T lymphocytes (CD8⁺ cells) releasing the toxic contents of their lytic granules at the IS formed with MHC-I-bearing cell targets for specific killing (Hivroz et al., 2012; de la Roche et al., 2016). Directional vesicular trafficking is, however, also the main mechanism by which T lymphocytes ensure the continuous availability of a functional pool of TCRs at the IS to sustain signaling during cell activation (Soares et al., 2013; Onnis et al., 2016; Finetti et al., 2017). This is achieved through the delivery to the synaptic membrane of TCRs associated with a pool of endosomes that undergo polarized recycling in a process regulated by both the tubulin and actin cytoskeletons (Martín-Cófreces and Sánchez-Madrid, 2018; Mastrogiovanni et al., 2020).

The striking architecture of the IS was described over 20 years ago. Not surprisingly, rearrangements of TCRs, adhesion molecules, costimulatory receptors, and membrane-associated signaling mediators occurring at the region of the T-cell plasma membrane at the contact with cognate APC have been extensively investigated. Only more recently has vesicular trafficking entered the picture with the finding that intracellular pools of the TCR and other components of the IS play a key role in this process beyond the known function of polarized delivery of effectors in differentiated T cells. From the initial finding that intracellular TCRs are delivered to the synaptic membrane through polarized recycling, it has become clear that the TCR is by no means unique in this respect. A wide array of other membrane-associated molecules, including receptors such as CD28 and signaling mediators such as Lck and LAT, have been demonstrated to undergo polarized recycling (Onnis and Baldari, 2019). Strikingly, with the identification of new regulators of the traffic of these molecules, achieved with essential input from the fields of vesicular trafficking and ciliogenesis (Cassioli and Baldari, 2019), it is now clear that a diversity of recycling pathways characterized by unique combinations of Rab GTPases and respective GEFs, v- and t-SNAREs, and tethering proteins coexist within the classical recycling pathways defined by Rab11 (Onnis et al., 2016).

While a role for microtubules and microtubule motors for the movement of these endosomes was expected, the identification of endosomal F-actin as a key player in the sorting of recycling molecules and their coupling to microtubules has brought a new layer of complexity to the process of polarized recycling to the IS. The identification of the retromer, retriever, and the CCC complex as a different means to achieve the polymerization of new actin filaments at endosome subdomains enriched in specific receptors that are destined for recycling has highlighted a diversity also at this step of the pathway (Wang et al., 2018; Simonetti and Cullen, 2019). Further work will be required to unravel the mechanisms and molecular machineries responsible for the specificity in the selection of both the receptors to be sorted for recycling and the respective actin-nucleating complex on which their transit from early to recycling endosomes depends. Additionally, how membrane subdomains are generated at early endosomes to serve as hubs for the accumulation of individual receptors and associated regulators remains elusive, as do the fine details of local force generation for the abscission of vesicles that will mature to recycling endosomes. We expect that a multidisciplinary approach to these questions capitalizing on converging new knowledge and new technologies gleaned from immunology, cell biology, and biophysics will be crucial to unravel the increasing complexity of the process of IS assembly.

NC and CB wrote the manuscript. NC prepared the artwork. Both the authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.