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The characterization of lipid binding to lipid transfer proteins (LTPs) is fundamental to understand their molecular mechanism. However, several structures of LTPs, and notably those proposed to act as bridges between membranes, do not provide the precise location of their endogenous lipid ligands. To address this limitation, computational approaches are a powerful alternative methodology, but they are often limited by the high flexibility of lipid substrates. Here, we develop a protocol based on unbiased coarse-grain molecular dynamics simulations in which lipids placed away from the protein can spontaneously bind to LTPs. This approach accurately determines binding pockets in LTPs and provides a working hypothesis for the lipid entry pathway. We apply this approach to characterize lipid binding to bridge LTPs of the Vps13-Atg2 family, for which the lipid localization inside the protein is currently unknown. Overall, our work paves the way to determine binding pockets and entry pathways for several LTPs in an inexpensive, fast, and accurate manner., (© 2024 Srinivasan et al.)

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The exocyst is a hetero-octameric complex involved in the exocytosis arm of cellular trafficking. Specifically, it tethers secretory vesicles to the plasma membrane, but it is also a main convergence point for many players of exocytosis: regulatory proteins, motor proteins, lipids and Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor (SNARE) proteins are all connected physically by the exocyst. Despite extensive knowledge about its structure and interactions, the exocyst remains an enigma precisely because of its increasingly broad and flexible role across the exocytosis process. To solve the molecular mechanism of such a multi-tasking complex, dynamical structures with self, other proteins, and environment should be described. And to do this, interrogation within contexts increasingly close to native conditions is needed. Here we provide a perspective on how different experimental contexts have been used to study the exocyst, and those that could be used in the future. This review describes the structural breakthroughs on the isolated in vitro exocyst, followed by the use of membrane reconstitution assays for revealing in vitro exocyst functionality. Next, it moves to in situ cell contexts, reviewing imaging techniques that have been, and that ideally could be, used to look for near-native structure and organization dynamics. Finally, it looks at the exocyst structure in situ within evolutionary contexts, and the potential of structure prediction therein. From in vitro, to in situ, cross-context investigation of exocyst structure has begun, and will be critical for functional mechanism elucidation., (© 2024 The Author(s).)

The acetylation of autophagy protein 9 A (ATG9A) in the lumen of the endoplasmic reticulum (ER) by ATase1 and ATase2 regulates the induction of reticulophagy. Analysis of the ER-specific ATG9A interactome identified calreticulin (CALR), an ER luminal Ca +2 -binding chaperone, as key for ATG9A activity. Specifically, if acetylated, ATG9A is sequestered by CALR and prevented from engaging FAM134B and SEC62. Under this condition, ATG9A is unable to activate the autophagy core machinery. In contrast, when non-acetylated, ATG9A is released by CALR and able to engage FAM134B and SEC62. In this study, we report that Ca +2 dynamics across the ER membrane regulate the ATG9A-CALR interaction as well as the ability of ATG9A to trigger reticulophagy. We show that the Ca +2 -binding sites situated on the C-domain of CALR are essential for the ATG9A-CALR interaction. Finally, we show that K359 and K363 on ATG9A can influence the ATG9A-CALR interaction. Collectively, our results disclose a previously unidentified aspect of the complex mechanisms that regulate ATG9A activity. They also offer a possible area of intersection between Ca +2 metabolism, acetyl-CoA metabolism, and ER proteostasis., (© 2024. The Author(s).)

In eukaryotes, integration of sterols into the vacuolar/lysosomal membrane is critically dependent on the Niemann-Pick type C (NPC) system. The system consists of an integral membrane protein, called NCR1 in yeast, and NPC2, a luminal soluble protein that transfers sterols to the N-terminal domain (NTD) of NCR1 before membrane integration. Both proteins have been implicated in sterol homeostasis of yeast and humans. Here, we investigate sterol and lipid binding of the NCR1/NPC2 transport system and determine crystal structures of the sterol binding NTD. The NTD binds both ergosterol and cholesterol, with nearly identical conformations of the binding pocket. Apart from sterols, the NTD can also bind fluorescent analogs of phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, as well as sphingosine and ceramide. We confirm the multi-lipid scope of the NCR1/NPC2 system using photo-crosslinkable and clickable lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pac-ceramide. Finally, we reconstitute the transfer of pac-sphingosine from NPC2 to the NTD in vitro. Collectively, our results support that the yeast NPC system can work as versatile machinery for vacuolar homeostasis of structurally diverse lipids, besides ergosterol., (© 2024 Nel et al.)

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have achieved impressive antitumor clinical outcomes. However, the limited response rates suggest the incomplete understanding of PD-L1 regulation. Here, we demonstrate that vacuole protein sorting 11 and 18 (VPS11/18), two key players in vesicular trafficking, positively regulate PD-L1 and confer resistance to immune checkpoint blockade therapy. VPS11/18 interact with PD-L1 in endosome recycling accompanied by promoting PD-L1 glycosylation and protein stability. VPS18 deficiency enhances antitumor immune response. Pharmacological inhibition by VPS18 inhibitor RDN impaired PD-L1 member trafficking and protein stability. Combination treatment of RDN and anti-cytotoxic T lymphocyte-associated antigen 4 synergistically enhances antitumor efficacy in aggressive and drug-resistant tumors. RDN exerted lung-preferred distribution and good bioavailability, suggesting a favorable drug efficacy. Together, our study links VPS18/11-mediated trans-Golgi network recycling of PD-L1 and points to a promising treatment strategy for the enhancement of antitumor immunity.

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing neuron activities or supporting network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif "SAYV" mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system., (© 2024. The Author(s).)

VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB (Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis., (© 2024. The Author(s).)

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To clarify the effect of growth and advancing age on lymphatic capillarization in rat skeletal muscles, we examined the histological and biochemical changes of lymphatic capillaries in different fiber types of skeletal muscles across juvenile, young, and middle-aged generations., Methods: We collected the tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) muscles. Immunohistochemical staining using LYVE-1 and CD31 markers was used for lymphatic and blood capillaries, respectively. Real-time PCR was used to analyze mRNA expression of lymphangiogenic factors., Results: The density of LYVE-1-positive lymphatic capillaries in the muscles peaked during the juvenile period and subsequently decreased with increasing age. In contrast to blood capillaries, fast-twitch dominant muscles (i.e., TA and EDL) exhibited an age-related decrease in lymphatic capillaries. Similar to blood capillaries, lymphatic capillaries were abundant in SOL, a slow-twitch dominant muscle, which showed less susceptibility to age-related lymphatic decline. The mRNA expression of lymphangiogenic factors was significantly upregulated in SOL and decreased in all muscles of middle-aged rats., Conclusions: The age-related decrease of lymphatic capillaries in fast-twitch muscles might be associated with age-related muscle atrophy., (© 2024 John Wiley & Sons Ltd.)

Type IV P-type ATPases (P4-ATPases) are lipid flippases that generate an asymmetric membrane organization essential for cell viability. The five budding yeast P4-ATPases traffic between the Golgi complex, plasma membrane, and endosomes but how they are recycled from the endolysosomal system to the Golgi complex is poorly understood. In this study, we find that P4-ATPase endosomal recycling is primarily driven by the retromer complex and the F-box protein Rcy1. Defects in P4-ATPase recycling result in their mislocalization to the vacuole and a substantial loss of membrane asymmetry. The P4-ATPases contain multiple predicted retromer sorting signals, and the characterization of these signals in Dnf1 and Dnf2 led to the identification of a novel retromer-dependent signal, IPM[ST] that acts redundantly with predicted motifs. Together, these results emphasize the importance of endosomal recycling for the functional localization of P4-ATPases and membrane organization., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Metastasis, the primary cause of death in lung cancer patients, is facilitated by cytoskeleton remodeling, which plays a crucial role in cancer cell migration and invasion. However, the precise regulatory mechanisms of intracellular trafficking proteins involved in cytoskeleton remodeling remain unclear. In this study, we have identified Rabenosyn-5 (Rbsn) as an inhibitor of filopodia formation and lung cancer metastasis. Mechanistically, Rbsn interacts with CDC42 and functions as a GTPase activating protein (GAP), thereby inhibiting CDC42 activity and subsequent filopodia formation. Furthermore, we have discovered that Akt phosphorylates Rbsn at the Thr253 site, and this phosphorylation negates the inhibitory effect of Rbsn on CDC42 activity. Additionally, our analysis reveals that Rbsn expression is significantly downregulated in lung cancer, and this decrease is associated with a worse prognosis. These findings provide strong evidence supporting the role of Rbsn in suppressing lung cancer progression through the inhibition of metastasis., (© 2024. The Author(s).)

Endosomes are central protein-sorting stations at the crossroads of numerous membrane trafficking pathways in all eukaryotes. They have a key role in protein homeostasis and cellular signalling and are involved in the pathogenesis of numerous diseases. Endosome-associated protein assemblies or coats collect transmembrane cargo proteins and concentrate them into retrieval domains. These domains can extend into tubular carriers, which then pinch off from the endosomal membrane and deliver the cargoes to appropriate subcellular compartments. Here we discuss novel insights into the structure of a number of tubular membrane coats that mediate the recruitment of cargoes into these carriers, focusing on sorting nexin-based coats such as Retromer, Commander and ESCPE-1. We summarize current and emerging views of how selective tubular endosomal carriers form and detach from endosomes by fission, highlighting structural aspects, conceptual challenges and open questions., (© 2024. Springer Nature Limited.)

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41 , core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases. Abbreviations : CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.

Objectives: Uncertainties remain regarding the effect of elevated glucose levels on lymphatic metastasis of cancer cells. Our study elucidated the mechanisms linking high glucose to lymphangiogenesis and lymphatic barrier-related factors and investigated the protective role of linagliptin against lymphatic barrier dysfunction., Materials and Methods: A CAL-27-LEC co-culture system was established. Sodium fluorescein permeability assay observed lymphatic endothelial cell permeability. Western blotting and RT-qPCR detected protein and mRNA expression under different conditions, respectively. CCK-8, scratch wound healing, and transwell assays revealed cell migration and proliferation. Tube formation experiment tested capacity for endothelial tube formation. Immunohistochemical staining analyzed tissue sections from 43 oral cancer individuals with/without diabetes., Results: In high-glucose co-culture system, we observed increased lymphatic barrier permeability and decreased expression of ZO-1 and occludin, two tight-junction proteins; conversely, the expression of PAR2, a high permeability-related protein, was increased. Following linagliptin treatment, the expression levels of VEGF-C, VEGFR-3, and PAR2 decreased, while those of ZO-1 and occludin increased. Considerably higher levels of LYVE-1 expression in individuals with diabetes than in those without diabetes., Conclusions: By ameliorating the high glucose-induced disruption of the lymphatic endothelial barrier, linagliptin may reduce lymphangiogenesis and exhibit an inhibitory effect on lymphatic metastasis in oral cancer patients with diabetes., (© 2024 Wiley Periodicals LLC.)

The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal "membrane trafficking" as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action., (© 2024. The Author(s).)

Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal disease in swine, for which no antiviral drugs or vaccines are currently available. Studying viral-host protein-protein interactions advances our understanding of the molecular mechanisms underlying viral replication and pathogenesis and can facilitate the discovery of antiviral therapeutics. In this study, we employed affinity tagging and purification mass spectrometry to characterize the interactome of VPS39, an important cellular factor during the early phase of ASFV replication. The interaction network of VPS39 revealed associations with mitochondrial proteins involved in membrane contact sites formation and cellular respiration. We show that the ASFV proteins CP204L and A137R target VPS39 by interacting with its clathrin heavy-chain functional domain. Furthermore, we elaborate on the potential mechanisms by which VPS39 may contribute to ASFV replication and prioritize interactions for further investigation into mitochondrial protein function in the context of ASFV infection.

In this issue of Developmental Cell, Jiang et al. report that the Arabidopsis HOPS tethering complex subunit VPS41 acts to catalyze the formation of a degradation pathway composed of a hybrid of autophagosomes and late endosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Several years ago, dozens of cases were described in patients with symptoms very similar to mucopolysaccharidosis (MPS). This new disease entity was described as mucopolysaccharidosis-plus syndrome (MPSPS). The name of the disease indicates that in addition to the typical symptoms of conventional MPS, patients develop other features such as congenital heart defects and kidney and hematopoietic system disorders. The symptoms are highly advanced, and patients usually do not survive past the second year of life. MPSPS is inherited in an autosomal recessive manner and is caused by a homozygous-specific mutation in the gene encoding the VPS33A protein. To date, it has been described in 41 patients. Patients with MPSPS exhibited excessive excretion of glycosaminoglycans (GAGs) in the urine and exceptionally high levels of heparan sulfate in the plasma, but the accumulation of substrates is not caused by a decrease in the activity of any lysosomal enzymes. Here, we discuss the pathomechanisms and symptoms of MPSPS, comparing them to those of MPS. Moreover, we asked the question whether MPSPS should be classified as a type of MPS or a separate disease, as contrary to 'classical' MPS types, despite GAG accumulation, no defects in lysosomal enzymes responsible for degradation of these compounds could be detected in MPSPS. The molecular mechanism of the appearance of GAG accumulation in MPSPS is suggested on the basis of results available in the literature.

ATG9A is the only integral membrane protein among core autophagy-related (ATG) proteins. We previously found that ATG9A does not co-assemble into synaptophysin-positive vesicles, but rather, localizes to a distinct pool of vesicles within synapsin condensates in both fibroblasts and nerve terminals. The endocytic origin of these vesicles further suggests the existence of different intracellular sorting or segregation mechanisms for ATG9A and synaptophysin in cells. However, the precise underlying mechanism remains largely unknown. In this follow-up study, we investigated the endosomal localization of these two proteins by exploiting the advantages of a Rab5 mutant that induces the formation of enlarged endosomes. Notably, ATG9A and synaptophysin intermix perfectly and do not segregate on giant endosomes, indicating that the separation of these two proteins is not solely caused by the inherent properties of the proteins, but possibly by other unknown factors., (© 2024. The Author(s).)

Vacuolar protein sorting 35 (VPS35), a critical component of the retromer complex, plays a pivotal role in the pathogenesis of neurodegenerative diseases (NDs). It is involved in protein transmembrane sorting, facilitating the transport from endosomes to the trans-Golgi network (TGN) and plasma membrane. Recent investigations have compellingly associated mutations in the VPS35 gene with neurodegenerative disorders such as Parkinson's and Alzheimer's disease. These genetic alterations are implicated in protein misfolding, disrupted autophagic processes, mitochondrial dysregulation, and synaptic impairment. Furthermore, VPS35 exerts a notable impact on neurogenesis by influencing neuronal functionality, protein conveyance, and synaptic performance. Dysregulation or mutation of VPS35 may escalate the progression of neurodegenerative conditions, underscoring its pivotal role in safeguarding neuronal integrity. This review comprehensively discusses the role of VPS35 and its functional impairments in NDs. Furthermore, we provide an overview of the impact of VPS35 on neurogenesis and further explore the intricate relationship between neurogenesis and NDs. These research advancements offer novel perspectives and valuable insights for identifying potential therapeutic targets in the treatment of NDs., (© 2024 International Society for Neurochemistry.)

Mutations in the human creatine transporter 1 (CRT1/SLC6A8) cause the creatine transporter deficiency syndrome, which is characterized by intellectual disability, epilepsy, autism, and developmental delay. The vast majority of mutations cause protein misfolding and hence reduce cell surface expression. Hence, it is important to understand the molecular machinery supporting folding and export of CRT1 from the endoplasmic reticulum (ER). All other SLC6 members thus far investigated rely on a C-terminal motif for binding the COPII-component SEC24 to drive their ER export; their N-termini are dispensable. Here, we show that, in contrast, in CRT1 the C-terminal ER-export motif is cryptic and it is the N-terminus, which supports ER export. This conclusion is based on the following observations: (i) siRNA-induced depletion of individual SEC24 isoforms revealed that CRT1 relied on SEC24C for ER export. However, mutations of the C-terminal canonical ER-export motif of CRT1 did not impair its cell surface delivery. (ii) Nevertheless, the C-terminal motif of CRT1 was operational in a chimeric protein comprising the serotonin transporter (SERT/SLC6A4) and the C-terminus of CRT1. (iii) Tagging of the N-terminus-but not the C-terminus-with yellow fluorescent protein (YFP) resulted in ER retention. (iv) Serial truncations of the N-terminus showed that removal of ≥51 residues of CRT1 impaired surface delivery, because the truncated CRT1 were confined to the ER. (v) Mutation of P51 to alanine also reduced cell surface delivery of CRT1 and relieved its dependence on SEC24C. Thus, the ER-export motif in the N-terminus of CRT1 overrides the canonical C-terminal motif., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment (TME) and can induce functional polarization of tumor macrophages. This study aimed to explore the effect of CAFs-derived exosome LINC01833 on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells and its mechanism. Tumor tissues (n = 3) and adjacent noncancerous tissues (n = 3) were collected from patients with NSCLC, and fibroblasts (CAF, NF) were isolated from the two tissues. Expression of LINC01833/miR-335-5p/VAPA in NSCLC clinical tissues and cell lines was detected by RT-qPCR. Exosomes of CAFs and NFs were isolated by ultracentrifugation. Cell proliferation, migration, invasion, and M2 macrophage polarization were detected by MTT, transwell, wound-healing assay, and flow cytometry assay, while western blot was used to verify the expression of M2 macrophage polarization-related proteins. Tumor volume weight and M2 macrophage polarization were detected by tumor xenografts in nude mice. LINC01833 was highly expressed in NSCLC tumor tissues and cells. Knockdown of LINC01833 exosomes could significantly inhibit proliferation, migration, invasion of NSCLC cells, and M2 macrophage polarization of THP-1 cells, while simultaneous knockdown of miR-335-5p on the above basis could reverse the effect of knockdown of LINC01833. In vivo experiments also indicated that knockdown of LINC01833 exosomes suppressed tumor growth and M2 macrophage polarization. CAF-derived LINC01833 exosomes can promote the proliferation, migration and invasion of NSCLC cells and M2 macrophage polarization by inhibiting miR-335-5p and regulating VAPA activity., (© 2024 Wiley Periodicals LLC.)

VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome., (© 2024 Du et al.)

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here, we show that VPS13B is localized at the interface between proximal and distal Golgi subcompartments and that Golgi complex reformation after Brefeldin A (BFA)-induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by the loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b ortholog, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in the dynamics of Golgi membranes and that VPS13B may be assisted in this function by FAM177A1., (© 2024 Ugur et al.)

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson's disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1 , GPNMB , LRRK2 , PINK1 , PRKN , SNCA , VPS13C , and VPS35 . Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1 , being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae , can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1 . This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

Mitochondrial population maintenance in neurons is essential for neuron function and survival. Contact sites between mitochondria and the endoplasmic reticulum (ER) are poised to regulate mitochondrial homeostasis in neurons. These contact sites can facilitate transfer of calcium and lipids between the organelles and have been shown to regulate aspects of mitochondrial dynamics. Vesicle-associated membrane protein-associated protein B (VapB) is an ER membrane protein present at a subset of ER-mitochondrial contact sites. A proline-to-serine mutation in VapB at amino acid 56 (P56S) correlates with susceptibility to amyotrophic lateral sclerosis (ALS) type 8. Given the relationship between failed mitochondrial health and neurodegenerative disease, we investigated the function of VapB in mitochondrial population maintenance. We demonstrated that transgenic expression of VapB P56S in zebrafish larvae (sex undetermined) increased mitochondrial biogenesis, causing increased mitochondrial population size in the axon terminal. Expression of wild-type VapB did not alter biogenesis but, instead, increased mitophagy in the axon terminal. Using genetic manipulations to independently increase mitochondrial biogenesis, we show that biogenesis is normally balanced by mitophagy to maintain a constant mitochondrial population size. VapB P56S transgenics fail to increase mitophagy to compensate for the increase in mitochondrial biogenesis, suggesting an impaired mitophagic response. Finally, using a synthetic ER-mitochondrial tether, we show that VapB's function in mitochondrial turnover is likely independent of ER-mitochondrial tethering by contact sites. Our findings demonstrate that VapB can control mitochondrial turnover in the axon terminal, and this function is altered by the P56S ALS-linked mutation., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are highly conserved endoplasmic reticulum (ER)-resident proteins that establish ER contacts with multiple membrane compartments in many eukaryotes. However, VAP-mediated membrane-tethering mechanisms remain ambiguous. Here, focusing on fission yeast ER-plasma membrane (PM) contact formation, using systematic interactome analyses and quantitative microscopy, we predict a non-VAP-protein direct binding-based ER-PM coupling. We further reveal that VAP-anionic phospholipid interactions may underlie ER-PM association and define the pH-responsive nature of VAP-tethered membrane contacts. Such conserved interactions with anionic phospholipids are generally defective in amyotrophic lateral sclerosis-associated human VAPB mutant. Moreover, we identify a conserved FFAT-like motif locating at the autoinhibitory hotspot of the essential PM proton pump Pma1. This modulatory VAP-Pma1 interaction appears crucial for pH homeostasis. We thus propose an ingenious strategy for maintaining intracellular pH by coupling Pma1 modulation with pH-sensory ER-PM contacts via VAP-mediated interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exo70, a key exocyst complex component, is crucial for cell motility and extracellular matrix (ECM) remodeling in cancer metastasis. Despite its potential as a drug target, Exo70's post-translational modifications (PTMs) are poorly characterized. Here, we report that Exo70 is transamidated on Gln5 with Lys56 of cystatin A by transglutaminases TGM1 and TGM3, promoting tumor metastasis. This modification enhances Exo70's association with other exocyst subunits, essential for secreting matrix metalloproteinases, forming invadopodia, and delivering integrins to the leading edge. Tumor suppressor liver kinase B1 (LKB1), whose inactivation accelerates metastasis, phosphorylates TGM1 and TGM3 at Thr386 and Thr282, respectively, to inhibit their interaction with Exo70 and the following transamidation. Cantharidin, a US Food and Drug Administration (FDA)-approved drug, inhibits Exo70 transamidation to restrain tumor cell migration and invasion. Together, our findings highlight Exo70 transamidation as a key molecular mechanism and target and propose cantharidin as a therapeutic strategy with direct clinical translational value for metastatic cancers, especially those with LKB1 loss., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains., Competing Interests: Competing interests statement:The authors declare no competing interest.

The retromer is a cellular structure that recruits and recycles proteins inside the cell. In mammalian and yeast, the retromer components have been widely studied, but very little in parasites. In yeast, it is formed by a SNX-BAR membrane remodeling heterodimer and the cargo selecting complex (CSC), composed by three proteins. One of them, the Vps26 protein, possesses a flexible and intrinsically disordered region (IDR), that facilitates interactions with other proteins and contributes to the retromer binding to the endosomal membrane. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, the retromer actively participates during the high mobility and phagocytosis of trophozoites, but the molecular details in these events, are almost unknown. Here, we studied the EhVps26 role in phagocytosis. Bioinformatic analyses of EhVps26 revealed a typical arrestin folding structure of the protein, and a long and charged IDR, as described in other systems. EhVps26 molecular dynamics simulations (MDS) allowed us to predict binding pockets for EhVps35, EhSNX3, and a PX domain-containing protein; these pockets were disorganized in a EhVps26 truncated version lacking the IDR. The AlphaFold2 software predicted the interaction of EhVps26 with EhVps35, EhVps29 and EhSNX3, in a model similar to the reported mammalian crystals. By confocal and transmission electron microscopy, EhVps26 was found in the trophozoites plasma membrane, cytosol, endosomes, and Golgi-like apparatus. During phagocytosis, it followed the erythrocytes pathway, probably participating in cargoes selection and recycling. Ehvps26 gene knocking down evidenced that the EhVps26 protein is necessary for efficient phagocytosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Martínez-Valencia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Endoplasmic reticulum (ER)-endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER-endolysosome membrane contact sites mediate the ER-endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP-ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER-endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER-endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.

Export of secretory cargoes from the endoplasmic reticulum (ER) requires COPII proteins, which were first identified for their ability to coat small vesicles that bud from the ER. Recent data indicate that COPII proteins can also organize into a collar at the necks of tubules, as well as phase-separate into liquid-like condensates. Thus, COPII assemblies seem to be tailored to accommodate variations in the size and quantities of cargo secreted., (© 2024 Malhotra.)

EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Introduction: Vacuolar Protein Sorting 35 (VPS35) is pivotal in the retromer complex, governing transmembrane protein trafficking within cells, and its dysfunction is implicated in neurodegenerative diseases. A missense mutation, Asp620Asn (D620N), specifically ties to familial late-onset Parkinson's, while reduced VPS35 levels are observed in Alzheimer's, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and tauopathies. VPS35's absence in certain neurons during development can initiate neurodegeneration, highlighting its necessity for neural health. Present therapeutic research mainly targets the clearance of harmful protein aggregates and symptom management. Innovative treatments focusing on VPS35 are under investigation, although fully understanding the mechanisms and optimal targeting strategies remain a challenge., Areas Covered: This review offers a detailed account of VPS35's discovery, its role in neurodegenerative mechanisms - especially in Parkinson's and Alzheimer's - and its link to other disorders. It shines alight on recent insights into VPS35's function in development, disease, and as a therapeutic target., Expert Opinion: VPS35 is integral to cellular function and disease association, making it a significant candidate for developing therapies. Progress in modulating VPS35's activity may lead to breakthrough treatments that not only slow disease progression but may also act as biomarkers for neurodegeneration risk, marking a step forward in managing these complex conditions.

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-β bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Most cancers and in particular carcinomas metastasise via the lymphatics to draining lymph nodes from where they can potentially achieve systemic dissemination by invasion of high endothelial blood venules (HEVs) in the paracortex [1, 2]. Currently however, the mechanisms by which tumours invade and migrate within the lymphatics are incompletely understood, although it seems likely they exploit at least some of the normal physiological mechanisms used by immune cells to access lymphatic capillaries and traffic to draining lymph nodes in the course of immune surveillance, immune modulation and the resolution of inflammation [3, 4]. Typically these include directional guidance via chemotaxis, haptotaxis and durotaxis, adhesion to the vessel surface via receptors including integrins, and junctional re-modelling by MMPs (Matrix MetalloProteinases) and ADAMs (A Disintegrin And Metalloproteinases) [5-7]. This short review focusses on a newly emerging mechanism for lymphatic entry that involves the large polysaccharide hyaluronan (HA) and its key lymphatic and immune cell receptors respectively LYVE-1 (Lymphatic Vessel Endothelial receptor) and CD44, and outlines recent work which indicates this axis may also be used by some tumours to aid nodal metastasis., (© 2023. The Author(s).)

Sterol regulatory element binding transcription factors (SREBPs) play a crucial role in lipid homeostasis. They are processed and transported to the nucleus via COPII, where they induce the expression of lipogenic genes. COPII maintains the homeostasis of organelles and plays an essential role in the protein secretion pathways in eukaryotes. The formation of COPII begins at endoplasmic reticulum exit sites (ERES), and is regulated by SEC16A, which provides a platform for the assembly of COPII. However, there have been few studies on the changes in SEC16A protein levels. The repetitive expansion of the hexanucleotide sequence GGGGCC within the chromosome 9 open reading frame 72 (C9orf72) gene is a prevalent factor in the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we found that the absence of C9orf72 leads to a decrease in SEC16A protein levels, resulting in reduced localization of the guanine nucleotide exchange factor SEC12 at the ERES. Consequently, the small GTP binding protein SAR1 is unable to bind the endoplasmic reticulum normally, impairing the assembly of COPII. Ultimately, the disruption of SREBPs transport decreases de novo lipogenesis. These results suggest that C9orf72 acts as a novel role in regulating lipid homeostasis and may serve as a potential therapeutic target for obesity., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared with control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export., Competing Interests: Conflict of interest: The authors declare no conflicts of interest.

Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH's colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH's EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy. Abbreviations : 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A 1 ; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl's balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.

Autophagy is a highly conserved catabolic mechanism by which unnecessary or dysfunctional cellular components are removed. The dysregulation of autophagy has been implicated in various neurodegenerative diseases, including Alzheimer's disease (AD). Understanding the molecular mechanism(s)/molecules that influence autophagy may provide important insights into developing therapeutic strategies against AD and other neurodegenerative disorders. Engulfment adaptor phosphotyrosine-binding domain-containing protein 1 (GULP1) is an adaptor that interacts with amyloid precursor protein (APP) to promote amyloid-β peptide production via an unidentified mechanism. Emerging evidence suggests that GULP1 has a role in autophagy. Here, we show that GULP1 is involved in autophagy through an interaction with autophagy-related 14 (ATG14), which is a regulator of autophagosome formation. GULP1 potentiated the stimulatory effect of ATG14 on autophagy by modulating class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1) activity. The effect of GULP1 is attenuated by a GULP1 mutation (GULP1m) that disrupts the GULP1-ATG14 interaction. Conversely, PI3KC3-C1 activity is enhanced in cells expressing APP but not in those expressing an APP mutant that does not bind GULP1, which suggests a role of GULP1-APP in regulating PI3KC3-C1 activity. Notably, GULP1 facilitates the targeting of ATG14 to the endoplasmic reticulum (ER). Moreover, the levels of both ATG14 and APP are elevated in the autophagic vacuoles (AVs) of cells expressing GULP1, but not in those expressing GULP1m. APP processing is markedly enhanced in cells co-expressing GULP1 and ATG14. Hence, GULP1 alters APP processing by promoting the entry of APP into AVs. In summary, we unveil a novel role of GULP1 in enhancing the targeting of ATG14 to the ER to stimulate autophagy and, consequently, APP processing., (© 2024. The Author(s).)

Respiratory syncytial virus (RSV) hijacks cholesterol or autophagy pathways to facilitate optimal replication. However, our understanding of the associated molecular mechanisms remains limited. Here, we show that RSV infection blocks cholesterol transport from lysosomes to the endoplasmic reticulum by downregulating the activity of lysosomal acid lipase, activates the SREBP2-LDLR axis, and promotes uptake and accumulation of exogenous cholesterol in lysosomes. High cholesterol levels impair the VAP-A-binding activity of ORP1L and promote the recruitment of dynein-dynactin, PLEKHM1, or HOPS VPS39 to Rab7-RILP, thereby facilitating minus-end transport of autophagosomes and autolysosome formation. Acidification inhibition and dysfunction of cholesterol-rich lysosomes impair autophagy flux by inhibiting autolysosome degradation, which promotes the accumulation of RSV fusion protein. RSV-F storage is nearly abolished after cholesterol depletion or knockdown of LDLR. Most importantly, the knockout of LDLR effectively inhibits RSV infection in vivo. These findings elucidate the molecular mechanism of how RSV co-regulates lysosomal cholesterol reprogramming and autophagy and reveal LDLR as a novel target for anti-RSV drug development., (© 2024. The Author(s).)

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod ∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases., (© 2024. The Author(s).)

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function., (© 2024. The Author(s).)

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.