EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

FAM91A1-TBC1D23 complex structure reveals human genetic variations susceptible for PCH.

Pontocerebellar hypoplasia (PCH) is a group of rare neurodevelopmental disorders with limited diagnostic and therapeutic options. Mutations in WDR11, a subunit of the FAM91A1 complex, have been found in patients with PCH-like symptoms; however, definitive evidence that the mutations are causal is still lacking. Here, we show that depletion of FAM91A1 results in developmental defects in zebrafish similar to that of TBC1D23, an established PCH gene. FAM91A1 and TBC1D23 directly interact with each other and cooperate to regulate endosome-to-Golgi trafficking of KIAA0319L, a protein known to regulate axonal growth. Crystal structure of the FAM91A1-TBC1D23 complex reveals that TBC1D23 binds to a conserved surface on FAM91A1 by assuming a Z-shaped conformation. More importantly, the interaction between FAM91A1 and TBC1D23 can be used to predict the risk of certain TBC1D23-associated mutations to PCH. Collectively, our study provides a molecular basis for the interaction between TBC1D23 and FAM91A1 and suggests that disrupted endosomal trafficking underlies multiple PCH subtypes.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells.

BACKGROUND: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. RESULTS: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. CONCLUSIONS: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum.

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20 , PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.

The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments.

For CD8 T lymphocytes to mount responses to cancer and virally-infected cells, dendritic cells must capture antigens present in tissues and display them as peptides bound to MHC-I molecules. This is most often accomplished through a pathway called antigen cross-presentation (XPT). Here, we report that the vesicular trafficking protein Rab39a is needed for optimal cross-presentation by dendritic cells in vitro and cross-priming of CD8 T cells in vivo. Without Rab39a, MHC-I presentation of intraphagosomal peptides is inhibited, indicating that Rab39a converts phagosomes into peptide-loading compartments. In this process, Rab39a promotes the delivery of MHC-I molecules from the endoplasmic reticulum (ER) to phagosomes, and increases the levels of peptide-empty MHC-I conformers that can be loaded with peptide in this compartment. Rab39a also increases the levels of Sec22b and NOX2, previously recognized to participate in cross-presentation, on phagosomes, thereby filling in a missing link into how phagosomes mature into cross-presenting vesicles.

Upregulated flotillins and sphingosine kinase 2 derail AXL vesicular traffic to promote epithelial-mesenchymal transition.

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

Aberrant post-translational modifications in endosomal trafficking are potential therapeutic targets to avert therapy resistance in solid cancers: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways.

Drugs targeting a single TK/RTK in the treatment of solid cancers has not had the same success seen in blood cancers. This is, in part, due to acquired resistance in solid cancers arising from a range of mechanisms including the upregulation of compensatory RTK signalling. Rather than attempting to inhibit individual compensatory RTK-requiring knowledge of which RTKs are upregulated in any given tumour-strategies to universally inhibit signalling from multiple RTKs may represent an effective alternative. Endosomal trafficking of RTKs is a common conduit that can regulate signalling from multiple RTKs simultaneously. As such, we posit that targeting endosomal trafficking-in particular, aberrant post-translational modifications in cancers that contribute to dysregulated endosomal trafficking-could inhibit oncogenic signalling driven by multiple RTKs and pave the way for the development of a novel class of inhibitors that shift the trafficking of RTKs to inhibit tumour growth.

UBA6 Inhibition Accelerates Lysosomal TRPML1 Depletion and Exosomal Secretion in Lung Cancer Cells.

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.

Formation of retromer transport carriers is disrupted by the Parkinson disease-linked Vps35 D620N variant.

Retromer core complex is an endosomal scaffold that plays a critical role in orchestrating protein trafficking within the endosomal system. Here we characterized the effect of the Parkinson's disease-linked Vps35 D620N in the endo-lysosomal system using Vps35 D620N rescue cell models. Vps35 D620N fully rescues the lysosomal and autophagy defects caused by retromer knock-out. Analogous to Vps35 knock out cells, the endosome-to-trans-Golgi network transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) is impaired in Vps35 D620N rescue cells because of a reduced capacity to form endosome transport carriers. Cells expressing the Vps35 D620N variant have altered endosomal morphology, resulting in smaller, rounder structures with less tubule-like branches. At the molecular level retromer incorporating Vps35 D620N variant has a decreased binding to retromer associated proteins wiskott-aldrich syndrome protein and SCAR homologue (WASH) and SNX3 which are known to associate with retromer to form the endosome transport carriers. Hence, the partial defects on retrograde protein trafficking carriers in the presence of Vps35 D620N represents an altered cellular state able to cause Parkinson's disease.

SORTING NEXIN2 proteins mediate stomatal movement and the response to drought stress by modulating trafficking and protein levels of the ABA exporter ABCG25.

The phytohormone abscisic acid (ABA) regulates ion channel activity and stomatal movement in response to drought stress. Cellular ABA levels change depending on cellular and environmental conditions via modulation of its biosynthesis, catabolism and transport. Although factors involved in ABA biosynthesis and degradation have been studied extensively, how ABA transporters are modulated to fine-tune ABA levels, especially under drought stress, remains elusive. Here, we show that Arabidopsis thaliana SORTING NEXIN 2 (SNX2) proteins play a critical role in endosomal trafficking of the ABA exporter ATP BINDING CASETTE G25 (ABCG25) via direct interaction at endosomes, leading to its degradation in the vacuole. In agreement, snx2a and snx2b mutant plants showed enhanced recycling of GFP-ABCG25 from early endosomes to the plasma membrane and higher accumulation of GFP-ABCG25. Phenotypically, snx2a and snx2b plants were highly sensitive to exogenous ABA and displayed enhanced ABA-mediated inhibition of inward K+ currents and ABA-mediated activation of slow anion currents in guard cells, resulting in an increased tolerance to drought stress. Based on these results, we propose that SNX2 proteins play a crucial role in stomatal movement and tolerance to drought stress by modulating the endosomal trafficking of ABCG25 and thus cellular ABA levels.

A glucose-starvation response governs endocytic trafficking and eisosomal retention of surface cargoes in budding yeast.

Eukaryotic cells adapt their metabolism to the extracellular environment. Downregulation of surface cargo proteins in response to nutrient stress reduces the burden of anabolic processes whilst elevating catabolic production in the lysosome. We show that glucose starvation in yeast triggers a transcriptional response that increases internalisation from the plasma membrane. Nuclear export of the Mig1 transcriptional repressor in response to glucose starvation increases levels of the Yap1801 and Yap1802 clathrin adaptors, which is sufficient to increase cargo internalisation. Beyond this, we show that glucose starvation results in Mig1-independent transcriptional upregulation of various eisosomal factors. These factors serve to sequester a portion of nutrient transporters at existing eisosomes, through the presence of Ygr130c and biochemical and biophysical changes in Pil1, allowing cells to persist throughout the starvation period and maximise nutrient uptake upon return to replete conditions. This provides a physiological benefit for cells to rapidly recover from glucose starvation. Collectively, this remodelling of the surface protein landscape during glucose starvation calibrates metabolism to available nutrients.This article has an associated First Person interview with the first author of the paper.

The kinesin motor Klp98A mediates apical to basal Wg transport.

Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.

PI3KC2ß inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2ß) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2ß in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2ß showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2ß in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2ß as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Truncating Mutations in UBAP1 Cause Hereditary Spastic Paraplegia.

The diagnostic gap for rare neurodegenerative diseases is still considerable, despite continuous advances in gene identification. Many novel Mendelian genes have only been identified in a few families worldwide. Here we report the identification of an autosomal-dominant gene for hereditary spastic paraplegia (HSP) in 10 families that are of diverse geographic origin and whose affected members all carry unique truncating changes in a circumscript region of UBAP1 (ubiquitin-associated protein 1). HSP is a neurodegenerative disease characterized by progressive lower-limb spasticity and weakness, as well as frequent bladder dysfunction. At least 40% of affected persons are currently undiagnosed after exome sequencing. We identified pathological truncating variants in UBAP1 in affected persons from Iran, USA, Germany, Canada, Spain, and Bulgarian Roma. The genetic support ranges from linkage in the largest family (LOD = 8.3) to three confirmed de novo mutations. We show that mRNA in the fibroblasts of affected individuals escapes nonsense-mediated decay and thus leads to the expression of truncated proteins; in addition, concentrations of the full-length protein are reduced in comparison to those in controls. This suggests either a dominant-negative effect or haploinsufficiency. UBAP1 links endosomal trafficking to the ubiquitination machinery pathways that have been previously implicated in HSPs, and UBAP1 provides a bridge toward a more unified pathophysiology.

Diversification of CORVET tethers facilitates transport complexity in Tetrahymena thermophila.

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.

Autophagy: a game changer for plant development and crop improvement.

MAIN CONCLUSION: Manipulation of autophagic pathway represents a tremendous opportunity for designing climate-smart crops with improved yield and better adaptability to changing environment. For exploiting autophagy to its full potential, identification and comprehensive characterization of adapters/receptor complex and elucidation of its regulatory network in crop plants is highly warranted.  Autophagy is a major intracellular trafficking pathway in eukaryotes involved in vacuolar degradation of cytoplasmic constituents, mis-folded proteins, and defective organelles. Under optimum conditions, autophagy operates at a basal level to maintain cellular homeostasis, but under stressed conditions, it is induced further to provide temporal stress relief. Our understanding of this highly dynamic process has evolved exponentially in the past few years with special reference to several plant-specific roles of autophagy. Here, we review the most recent advances in the field of autophagy in plants and discuss its potential implications in designing crops with improved stress and disease-tolerance, enhanced yield potential, and improved capabilities for producing metabolites of high economic value. We also assess the current knowledge gaps and the possible strategies to develop a robust module for biotechnological application of autophagy to enhance bioeconomy and sustainability of agriculture.

Unconventional p97/VCP-Mediated Endoplasmic Reticulum-to-Endosome Trafficking of a Retroviral Protein.

Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER) membrane into a functional N-terminal signal peptide (SP) and the C terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, and yet it was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was colocalized with Rab5-positive (Rab5+) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (containing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. IMPORTANCE Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.

Adeno-associated Virus 9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.

Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. IMPORTANCE There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.

Joining Forces for Cancer Treatment: From "TCR versus CAR" to "TCR and CAR".

T cell-based immunotherapy has demonstrated great therapeutic potential in recent decades, on the one hand, by using tumor-infiltrating lymphocytes (TILs) and, on the other hand, by engineering T cells to obtain anti-tumor specificities through the introduction of either engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Given the distinct design of both receptors and the type of antigen that is encountered, the requirements for proper antigen engagement and downstream signal transduction by TCRs and CARs differ. Synapse formation and signal transduction of CAR T cells, despite further refinement of CAR T cell designs, still do not fully recapitulate that of TCR T cells and might limit CAR T cell persistence and functionality. Thus, deep knowledge about the molecular differences in CAR and TCR T cell signaling would greatly advance the further optimization of CAR designs and elucidate under which circumstances a combination of both receptors would improve the functionality of T cells for cancer treatment. Herein, we provide a comprehensive review about similarities and differences by directly comparing the architecture, synapse formation and signaling of TCRs and CARs, highlighting the knowns and unknowns. In the second part of the review, we discuss the current status of combining CAR and TCR technologies, encouraging a change in perspective from "TCR versus CAR" to "TCR and CAR".