Import of proviral genome from the cytoplasm to the nucleus is a decisive step in the HIV-1 infection cycle, regulating which can decide the fate of HIV-1 infectivity. Exploring the heterogeneity in the replication potential of viruses emerging from various producer cells, we compared the infectivity dynamics of viruses produced by CD4+ T lymphocytes, a cell type that supports the HIV-1 propagation, with those from astrocytes that allow for limited viral replication. We found the viruses emerging from these two cell types not only differed in their infectivity, but also in the host proteins that get associated with these virions. We focused on Importin β1, an autonomous nuclear transport receptor, which was present in the virion fractions from CD4+ T lymphocytes but absent in those from astrocytes. Our analysis revealed that Importin β1 gets associated with the virus through interactions with HIV-1 Gag and Capsid proteins. Using Importin β1 knockout cell models, we found that virion-associated Importin β1 enhanced viral infectivity by facilitating the import of the viral Pre-integration complex (PIC) into the nucleus of an infected cell. Linking positive factors, such as Importin β1, to emerging virions can determine the viral infectivity in subsequent infection rounds, influencing disease progression.

In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g., IL1β, Acb1, FGF2) have been detected, and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on "Molecular Mechanisms and Physiology of Unconventional Secretion" is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.

Copper is one of the essential micronutrients utilized as a cofactor in a wide variety of biochemical reactions of metabolic pathways, including mitochondrial respiration and innate immune response. Cellular concentration and distribution of copper is regulated by copper-specific transporters, chaperones, metallothionein proteins and amino acids. Transcription of a major copper metallothionein, CUP1 is epigenetically regulated in Saccharomyces cerevisiae. Mutations in histones dysregulate cellular copper homeostasis due to abnormal epigenetic changes and cause diseases in humans, such as cancerous growth and neurological disorders. Low or higher cellular concentration of copper is associated with disorders such as Menkes and Wilson's disease, respectively. Higher concentrations of copper cause caspase-independent cell death known as cuproptosis and haemolytic anemia. We highlighted the existing knowledge regarding the significance of epigenetics and cellular factors in the regulation of copper metabolism and copper-regulated protein trafficking. We have also proposed a few future directions to explore the role of cellular pH dynamics, stoichiometry among metals, amino acids and protein metabolism, histone modifications, autophagy and mitochondrial respiration in regulating cellular copper metabolism. Altogether, we provide a comprehensive summary of cellular factors targeting copper metabolism for dissecting the underlying complex mechanism of copper dynamics in normal physiology and diseases.

Arf and Rab family small GTPases and their regulators, GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), play a central role in membrane trafficking. In this study, we focused on a recently reported GAP for Arf (and potentially Rab) proteins, the CSW complex, a part of a small family of longin domain-containing proteins that form complexes with GAP activity. This family also includes folliculin and GATOR1, which are GAPs for the Rag/Gtr GTPases. All three complexes are associated with lysosomes and play a role in nutrient signaling, the latter two being directly involved in the mTOR pathway. The role of CSW is not clear, but in addition to having GAP activity on Arf proteins in vitro, its mutation causes severe neurodegenerative diseases. Here we update the reported pan-eukaryotic presence of folliculin and GATOR1, and demonstrate that CSW is also found throughout eukaryotes, though with sporadic distribution. We identify highly conserved motifs in all CSW subunits, some shared with the catalytic subunits of folliculin and GATOR1, that provide new potential avenues for experimental exploration. Remarkably, one such conserved sequence, the "GP" motif, is also found in structurally related longin proteins present in the archaeal ancestor of eukaryotes.

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.

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.

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 and Arl15) 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.

The delivery of intracellular cargoes by kinesins is modulated at scales ranging from the geometry of the microtubule networks down to interactions with individual tubulins and their code. The complexity of the tubulin code and the difficulty in directly observing motor-tubulin interactions have hindered progress in pinpointing the precise mechanisms by which kinesin's function is modulated. As one such example, past experiments show that cleaving tubulin C-terminal tails (CTTs) lowers kinesin-1's processivity and velocity on microtubules, but how these CTTs intertwine with kinesin's processive cycle remains unclear. In this work, we formulate and interrogate several plausible mechanisms by which CTTs contribute to and modulate kinesin motion. Computational modeling bridges the gap between effective transport observations (processivity, velocities) and microscopic mechanisms. Ultimately, we find that a guiding mechanism can best explain the observed differences in processivity and velocity. Altogether, our work adds a new understanding of how the CTTs and their modulation via the tubulin code may steer intracellular traffic in both health and disease.

The secretion of insulin and glucagon by pancreatic β and α cells, respectively, is critical for glucose homeostasis. While the insulin granule dynamics are well-characterized, the intracellular behavior of glucagon secretory granules (GSG) remains poorly understood. Here, we analyze the mobility of GSGs in αTC1-9 cells and insulin secretory granules (ISG) in INS-1E cells using spatiotemporal correlation spectroscopy and single-particle tracking (SPT), with a focus on the role of the cytoskeleton in regulating their transport. Under basal conditions, SPT classification reveals that GSGs predominantly exhibit diffusive motion (57.6% ± 10%), with smaller fractions categorized as almost immobile (35.8% ± 10.6%) or drifted (6.6% ± 3%), closely resembling ISG dynamics. By disrupting microtubules, we confirmed their role as active tracks for directed granule transport in both cell types. Upon exposure to their respective secretory stimuli-high glucose for β cells and low glucose for α cells-both granule populations underwent a comparable shift toward increased diffusive and drifted motions. Treatment with the actin depolymerizing agent Latrunculin-B reproduced this stimulatory effect in INS-1E cells but not in αTC1-9 cells, suggesting that despite their overall similarity in granule behavior under physiological conditions, α and β cells may rely on partially distinct mechanisms to engage the cytoskeletal network.

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.

Cervical cancer (CC) exerts a considerable impact on women's health worldwide and presents persistent challenges to conventional therapeutic strategies due to its propensity for distant metastasis, postoperative recurrence, and variable drug resistance. Ferroptosis, a recently identified type of programmed cell death, offers promising potential for a therapeutic approach for CC. This paper reviews the regulatory processes involved in ferroptosis, including the sequential events leading to cell membrane rupture via lipid peroxidation and the changes in ferroptosis sensitivity as cervical cells progress from a healthy to a malignant condition. Additionally, the dynamic relationship between ferroptosis and CC transformation driven by high-risk HPV (HR-HPV) infection is examined, with a particular focus on how HR-HPV E6/E7 proteins influence ferroptosis sensitivity. By examining the factors associated with ferroptosis, this review provides insights into CC progression and prognosis. Furthermore, therapeutic strategies targeting ferroptosis are discussed, offering novel perspectives for effective treatment options for CC.

Among extracellular vesicles (EVs), exosomes, comprised within small EVs are bilayered nanovesicles carrying specific cargo that are released into the interstitial space in a highly regulated manner. In this study, we investigated the message transmitted through macrophage-derived small EVs in response to the interaction with Trypanosoma cruzi, the protozoan responsible for Chagas disease. We utilized two distinct parasite strains, the virulent CL Brener and the attenuated TCC. When taken up by naϊve macrophages (Mφs) in vitro, small EVs derived from TCC-infected cells favor an adverse environment for parasite spread, with M1-like cytokine pattern. In contrast, EVs from CL Brener-infected cells fostered a more permissive environment with reduced TNF-α/IL-10 ratio, higher phagocytic activity and reduced migration capacity, which may hinder a timely immune response. Further, while naïve Mφs' EVs induced iNOS and nitric oxide (NO) secretion, EVs from T. cruzi-infected Mφs failed to robustly activate iNOS, suggesting the parasite may modulate EV-mediated communication to avoid NO toxicity. In vivo assays showed distinct parasitemia courses with higher parasite burden when mice were treated with small EVs from CL Brener-infected Mφs. Overall, small EVs released by infected Mφs serve as messengers in T. cruzi infection, inducing different immune responses based on parasite virulence.

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.

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.

The mannose 6-phosphate (M6P) pathway is critical for lysosome biogenesis, facilitating the trafficking of hydrolases to lysosomes to ensure cellular degradative capacity. Fibroblast Growth Factor (FGF) signaling, a key regulator of skeletogenesis, has been linked to the autophagy-lysosomal pathway in chondrocytes, but its role in lysosome biogenesis remains poorly characterized. Here, using mass spectrometry, lysosome immune-purification, and functional assays, we reveal that RCS (Swarm rat chondrosarcoma cells) lacking FGF receptors 3 and 4 exhibit dysregulations of the M6P pathway, resulting in hypersecretion of lysosomal enzymes and impaired lysosomal function. We found that FGF receptors control the expression of M6P receptor genes in response to FGF stimulation and during cell cycle via the activation of the transcription factors TFEB and TFE3. Notably, restoring M6P pathway-either through gene expression or activation of TFEB-significantly rescues lysosomal defects in FGFR3;4-deficient RCS. These findings uncover a novel mechanism by which FGF signaling regulates lysosomal function, offering insights into the control of chondrocyte catabolism and the understanding of FGF-related human diseases.

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.

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.

Small extracellular vesicles (sEVs) originate from endosomes formed during cellular endocytosis, have a diameter ranging from 30 to 150 nm and are membrane-bound prior to release, sEVs may also be formed by budding of the plasma membrane to form ectosomes. sEVs transport proteins, RNA, microRNAs (miRNAs), DNA, and other bioactive substances to facilitate information exchange and may function as mediators under physiological conditions. sEVs have various pathological roles, especially when produced by tumor parenchyma and stromal cells for signaling in the tumor-induced microenvironment. The vesicles are considered potential tumor markers and there are broad prospects for developing tumor therapies by inhibiting sEV production, secretion and uptake and eliminating circulating sEVs. sEVs may be modified to deliver chemotherapeutic drugs and this approach has shown promising results for tumor inhibition and improved prognosis. The current study reviews the role of sEVs in tumor development and explores the potential for tumor treatment.

Recycling endosomes are essential for membrane trafficking, retrieving internalized cell surface receptors and lipids to the plasma membrane. In this study, we investigate the dynamics of tubular recycling endosomes (TREs) and their regulation. We demonstrate that TREs are highly dynamic structures that first undergo biogenesis and later fission upon internalization of CD98, a known clathrin-independent cargo. Our findings identify two new constituents and novel regulators of TRE function, CD2AP and CIN85, which are recruited to TRE through interactions with MICAL-L1 via their SH3 domains. Depletion of either CD2AP or CIN85 impairs recycling, demonstrating that these proteins play important roles in TRE function. Our study highlights the importance of coordinated protein interactions in maintaining endosomal function and identifies CD2AP and CIN85 as key regulators of the recycling pathway, potentially through their impact on the actin cytoskeleton. Understanding these mechanisms provides new insights into membrane trafficking and may have implications for diseases where endosomal recycling is disrupted.