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Human TENT5 family comprises four members (A-D) associated with different diseases of secretory cells. Homozygous mutations in TENT5A cause a rare form of osteogenesis imperfecta due to impaired collagen deposition by osteoblasts. TENT5C is frequently mutated or deleted in patients with multiple myeloma, the cancer of antibody-secreting plasma cells, and TENT5D alterations result in male infertility. TENT5 members are noncanonical poly(A)polymerases that selectively stabilize mRNAs encoding endoplasmic reticulum-imported proteins, thus promoting the expression of secretory cargoes and proteins involved in folding, glycosylation, and trafficking along the secretory apparatus. This specificity has been proposed to be linked to TENT5 localization at the membrane of the endoplasmic reticulum, thanks to their interaction with transmembrane FNDC3 proteins. Recently, key roles of TENT5 proteins have been described in cancer, bone homeostasis, immunity, stemness, and fertility. This review will comprehensively analyze the identified cellular functions of this novel family of secretory tuners in physiological and pathological conditions, highlighting the proposed molecular mechanisms and the remaining open questions.

This manuscript describes a novel unconventional secretory pathway that facilitates EGF receptor (EGFR) signaling from apical membranes in polarized epithelial cells responding to immune cell mediators. Epithelial tissues provide a physical barrier between our bodies and the external environment and share an intimate relationship with circulating and local immune cells. Our studies describe an unexpected connection between the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) and EGFR typically localized to basolateral membranes in polarized epithelial cells. These two molecules sit atop complex biological networks with a long history of shared investigative interest from the vantage point of signaling pathway interactions. We have discovered that TNF-α alters the functional landscape of fully polarized epithelial cells by changing the speed and direction of EGFR secretion. Our results show apical EGFR delivery occurs within minutes of de novo synthesis likely via a direct route from the endoplasmic reticulum without passage through the Golgi complex. Additionally, our studies have revealed that apical cellular compartmentalization constitutes an important mechanism to specify EGFR signaling via phosphatidylinositol-4,5-bisphosphate 3-kinase/protein-kinase-B pathways. Our study paves the way for a better understanding of how inflammatory cytokines fine-tune local homeostatic and inflammatory responses by altering the spatial organization of epithelial cell signaling systems.

In addition to the conventional endoplasmic reticulum (ER)-Golgi secretory pathway, alternative routes are increasingly recognized for their critical roles in exporting a growing number of secreted factors. These alternative processes, collectively referred to as unconventional protein secretion (UcPS), challenge traditional views of protein and membrane trafficking. Unlike the well-characterized molecular machinery of the conventional secretory pathway, the mechanisms underlying UcPS remain poorly understood. Various UcPS pathways may involve direct transport of cytosolic proteins across the plasma membrane or the incorporation of cargo proteins into intracellular compartments redirected for secretion. Identifying the specific chaperones, transporters and fusion machinery involved in UcPS cargo recognition, selection and transport is crucial to decipher how cargo proteins are selectively or synergistically directed through multiple secretory routes. These processes can vary depending on cell type and in response to particular stress conditions or cellular demands, underscoring the need for standardized tools and methods to study UcPS. Here, we combine the sensitivity of split NanoLuc Binary Technology with the versatility of the Retention Using Selective Hooks (RUSH) system to develop a straightforward and reliable cell-based assay for investigating both conventional and unconventional protein secretion. This system allows for the identification of intracellular compartments involved in UcPS cargo trafficking. Additionally, its sensitivity enabled us to demonstrate that disease-associated mutants or variants of Tau and superoxide dismutase-1 (SOD1) show altered secretion via UcPS. Finally, we leveraged this assay to screen for Alzheimer's disease risk factors, revealing a functional link between amyloid-beta production and Tau UcPS. This robust assay provides a powerful tool for increasing our knowledge of protein secretion mechanisms in physiological and pathological contexts.

The folded auto-inhibited state of kinesin-1 is stabilized by multiple weak interactions and binds poorly to microtubules. Here we investigate the extent to which homodimeric Drosophila kinesin-1 lacking light chains is activated by the dynein activating adaptor Drosophila BicD. We show that one or two kinesins can bind to the central region of BicD (CC2), a region distinct from that which binds dynein-dynactin (CC1) and cargo-adaptor proteins (CC3). Kinesin light chain significantly reduces the amount of kinesin bound to BicD and thus regulates this interaction. Binding of BicD to kinesin enhances processive motion, suggesting that the adaptor relieves kinesin auto-inhibition. In contrast, the kinesin-binding domain of microtubule-associated protein 7 (MAP7) has minimal impact on the fraction of motors moving processively while full-length MAP7 enhances kinesin-1 recruitment to the microtubule and run length because of its microtubule-binding domain. BicD thus relieves auto-inhibition of kinesin, while MAP7 enhances motor engagement with the microtubules. When BicD and MAP7 are combined, the most robust activation of kinesin-1 occurs, highlighting the crosstalk between adaptors and microtubule-associated proteins in regulating transport.

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Cell biological studies implicated Arl15 in regulating various cellular processes, including magnesium homeostasis and TGFβ signaling. However, the role of Arl15 in vesicular transport is poorly defined. We evaluated the function of Arl15 using techniques to quantify cargo trafficking to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization primarily to the Golgi and cell surface. The depletion of Arl15 causes the mislocalization of selective Golgi cargo, such as caveolin-2 and STX6, in the cells. Consistently, expression of GTPase-independent dominant negative mutants of Arl15 (Arl15^{V80A,A86L,E122K} and Arl15^C22Y,C23Y) results in mislocalization of caveolin-2 and STX6 from the Golgi. However, the localization of Arl15 to the Golgi is dependent on its palmitoylation and Arf1-dependent Golgi integrity. At the cellular level, Arl15 depleted cells display enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion compared to control cells. Collectively, these studies implicate a functional role for Arl15 in regulating cargo transport from the Golgi to regulate cell surface processes.

Modeling and simulation are transforming all fields of biology. Tools like AlphaFold have revolutionized structural biology, while molecular dynamics simulations provide invaluable insights into the behavior of macromolecules in solution or on membranes. In contrast, we lack effective tools to represent the dynamic behavior of the endomembrane system. Static diagrams that connect organelles with arrows are used to depict transport across space and time but fail to specify the underlying mechanisms. This static representation obscures the dynamism of intracellular traffic, freezing it in an immobilized framework. The intracellular transport of transferrin, a key process for cellular iron delivery, is among the best-characterized trafficking pathways. In this commentary, we revisit this process using a mathematical simulation of the endomembrane system. Our model reproduces many experimental observations and highlights the strong contrast between dynamic simulations and static illustrations. This commentary underscores the urgent need for a consensus-based minimal functional working model for the endomembrane system and emphasizes the importance of generating more quantitative experimental data-including precise measurements of organelle size, volume and transport kinetics-practices that were more common among cell biologists in past decades.

Intracellular pathogens rely on manipulating host endocytic pathways to ensure survival. Legionella and Chlamydia exploit host SNARE proteins, with Legionella cleaving syntaxin 17 (STX17) and Chlamydia interacting with VAMP8 and VAMP7. Similarly, Salmonella targets the host's endosomal fusion machinery, using SPI effectors like SipC and SipA to interact with syntaxin 6 (STX6) and syntaxin 8 (STX8), respectively, maintaining its vacuolar niche. Recent evidence highlights syntaxin 7 (STX7), a Qa-SNARE involved in endo-lysosomal fusion, as a potential Salmonella target. BioID screening revealed STX7 interactions with SPI-2 effectors SifA and SopD2, suggesting a critical role in Salmonella pathogenesis. We investigated the role of STX7 in Salmonella-containing vacuole (SCV) biogenesis and pathogenesis in macrophages and epithelial cells. Our findings indicate that STX7 levels and localization differ between these cell types during infection, reflecting the distinct survival strategies of Salmonella. Live cell imaging showed that STX7 is recruited to SCVs at different infection stages, with significantly altered distribution in HeLa cells at the late stage of infection. STX7 knockdown resulted in reduced bacterial survival, which was rescued upon overexpression of STX7 in both HeLa and RAW264.7 cells, suggesting Salmonella hijacks STX7 to evade lysosomal fusion and secure nutrients for intracellular replication. These results underscore the essential role of STX7 in maintaining SCVs and facilitating Salmonella survival. Further, the temporal expression of STX7 adaptor/binding partners in macrophages showed dynamic interactions with STX7 facilitating Salmonella infection and survival in host cells. Together, our study highlights STX7 as a critical host factor exploited by Salmonella, providing insights into the molecular mechanisms underlying its pathogenesis in macrophages and epithelial cells. These findings may inform strategies for targeting host-pathogen interactions to combat Salmonella infections.

In certain kinds of cells, clathrin-independently endocytosed cargo proteins are recycled back to the plasma membrane via specialized tubular-shaped endosomes, so-called tubular endosomes. Several regulators, including Rab small GTPases, have previously been reported to control tubular endosome structures, and one of the regulators, Rab22A, controls cargo sorting and tubule elongation. Since Rab activity is generally controlled by a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP), these upstream regulators would also be involved in tubular endosome formation. However, although we have previously reported that Vps9d1 is a Rab22A-GEF that controls tubular endosome formation, there have been no reports of Rab-GAPs that are required for tubular endosome formation. Here, we demonstrated by comprehensive screening of TBC/Rab-GAPs that four Rab-GAPs, TBC1D10B, TBC1D18, TBC1D22B and EVI5, are involved in tubular endosome formation in HeLa cells in a GAP-activity-dependent manner. Knockdown or overexpression of each of these Rab-GAPs resulted in the same phenotype, that is, reduced tubular endosome structures. Since one of these four Rab-GAPs, TBC1D10B, was able to reduce the amount of active Rab22A and the size of Rab22A-positive early endosomes, it is the most probable candidate for a Rab22A-GAP. Our findings suggest that a proper GTPase cycle is important for the control of tubular endosome formation.

The GARP complex is an evolutionarily conserved protein complex proposed to tether endosome-derived vesicles at the trans-Golgi network. While complete depletion of the GARP leads to severe trafficking and glycosylation defects, the primary defects linked to GARP dysfunction remain unclear. In this study, we utilized the mAID degron strategy to achieve rapid degradation of VPS54 in human cells, acutely disrupting GARP function. This resulted in the partial mislocalization and degradation of a subset of Golgi-resident proteins, including TGN46, ATP7A, TMEM87A, CPD, C1GALT1 and GS15. Enzyme recycling defects led to O-glycosylation abnormalities. Additionally, while fibronectin and cathepsin D secretion were altered, mannose-6-phosphate receptors were largely unaffected. Partial displacement of COPI, AP1 and GGA coats caused a significant accumulation of vesicle-like structures and large vacuoles. Electron microscopy detection of GARP-dependent vesicles and identifying specific cargo proteins provide direct experimental evidence of GARP's role as a vesicular tether. We conclude that the primary defects of GARP dysfunction involve vesicular coat mislocalization, accumulation of GARP-dependent vesicles, degradation and mislocalization of specific Golgi proteins and O-glycosylation defects.