Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-localized transmembrane protein. STING induces the type I interferon and inflammatory responses against a variety of double-stranded DNA (dsDNA) viruses, which is critical to limiting their infection and replication. In certain settings where self-DNAs (genomic or mitochondrial DNA) emerge in the cytosol or the intracellular membrane traffic is impaired, STING becomes activated and triggers inflammation, which may contribute to the pathogenesis of various autoinflammatory and neurodegenerative diseases including COPA syndrome and Parkinson's disease. The human STING gene holds genetic heterogeneity with R232, HAQ (R71H-G230A-R293Q), and H232 being the most common variants, and population stratification. A very recent study has shown that HAQ, not R232 or H232, mediates completely clinical protection in the pathogenesis of COPA syndrome. These results reveal, for the first time, the distinct activities of the major variants in the context of pathogenesis of autoinflammatory diseases. Besides these major variants, there exist minor pathogenic STING variants that cause an autoinflammatory disease called STING-associated vasculopathy with onset in infancy (SAVI). This review summarizes recent insights into human STING variants and their inflammatory activities.Key words: innate immunity, STING variants, COPA syndrome, membrane traffic, the Golgi.

Stimulator of interferon genes (STING) triggers the type I interferon and inflammatory responses against a variety of DNA pathogens, which is essential to limiting viral infection and replication. STING activates the downstream kinase TBK1 at the trans-Golgi network (TGN) and is degraded at lysosomes through a process called lysosomal microautophagy. Impaired STING targeting to lysosomes results in the prolonged inflammatory signal, which may be associated with a variety of neurodegenerative and autoinflammatory diseases. Thus, development of methods to quantify STING degradation helps understand the mechanism of lysosomal microautophagy and its related diseases. Here we report a quantitative method to monitor STING degradation with two luciferases, firefly luciferase (FLuc) and Nanoluciferase (NLuc). The expression plasmid is composed of FLuc, a P2A self-cleavage site, and NLuc-tagged STING. FLuc intensity reflects the total amount of translated protein, serving as an internal control, while NLuc intensity corresponds to the amount of STING. Comparison of the NLuc/FLuc ratio after STING stimulation reported the kinetics of decay of STING levels in live cells. This method should provide a useful complement to western blotting and fluorescence- activated cell sorter (FACS) analysis presently used to monitor STING degradation.Key words: Innate immunity, STING, membrane traffic, lysosomal degradation, luciferase.

Our body is constantly exposed to pathogens, and equipped with a highly elaborate immune system to fight against invading pathogens. The first line of defense is the innate immune system. It has evolved to detect conserved microbial molecular patterns, dubbed pathogen-associated molecular patterns (PAMPs), through pattern recognition receptors (PRRs). The binding of PRRs to PAMPs activates intracellular signalling cascades that lead to the expression of proinflammatory cytokines, type I interferons, and other antiviral proteins that all coordinate the elimination of pathogens and infected cells. PRRs can be classified as transmembrane receptors, including Toll-like receptors (TLRs) and some C-type lectin receptors (CLRs), and as cytosolic receptors including retinoic acid-inducible gene-I (RIG-I)-like receptors, nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, and cyclic GMP-AMP (cGAMP) synthase (cGAS). Studies have revealed that innate immune signals, including the ones activated by cytosolic PRRs, are triggered on organelle membranes. Here we review the recent insights into how organelle membranes and their associated membrane lipids contribute to PRR-mediated innate immune signals.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.

Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e., macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. In macroautophagy, a portion of the cytoplasm is wrapped by the autophagosome, which then fuses with lysosomes and delivers the engulfed cytoplasm for degradation. In CMA, the translocation of cytosolic substrates to the lysosomal lumen is directly across the limiting membrane of lysosomes. In microautophagy, lytic organelles, including endosomes or lysosomes, take up a portion of the cytoplasm directly. Although macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become evident that microautophagy plays a variety of cellular roles from yeast to mammals. Here we review the very recent updates of microautophagy. In particular, we focus on the feature of the degradative substrates and the molecular machinery that mediates microautophagy.

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from viruses or self-DNA from mitochondria/nuclei. Recently, gain-of-function mutations in STING have been identified in patients with STING-associated vasculopathy with onset in infancy (SAVI). The SAVI patients exhibit complex systemic vascular inflammation and interstitial lung disease, resulting in pulmonary fibrosis and respiratory failure. SAVI mouse models have recently developed, harbouring common SAVI mutations, such as N153S and V154M, which correspond to the human N154S and V155M, respectively. Interestingly, crosses of heterozygous SAVI mice did not yield homozygous SAVI mice as of embryonic day 14, indicating that homozygous SAVI embryos were not viable and that wild-type (WT) allele would function dominantly over SAVI alleles in terms of viability. However, the molecular mechanism underlying the dominance has not been understood. In the present study, we show that STING (WT) and STING (SAVI) can form heterocomplex. The heterocomplex localized primarily in the endoplasmic reticulum (ER) and failed to reach the trans-Golgi network (TGN), where STING activates the downstream kinase TBK1. SURF4 is the essential protein functioning in the retrieval of STING from the Golgi to the ER. The amount of SURF4 bound to STING (SAVI) significantly increased in the presence of STING (WT). These results suggest that STING (WT) can suppress the activity of STING (SAVI) by tethering STING (SAVI) to the ER through heterocomplex formation. The dormant heterocomplex formation may underlie, at least in part, the dominance of STING WT allele over SAVI alleles in the STING-triggered inflammatory response.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA or self-DNA leaked from mitochondria/nuclei. In response to the emergence of such DNAs in the cytosol, STING relocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase essential for the activation of STING-dependent downstream signalling. To understand at which subcellular compartments TBK1 becomes associated with STING, we generated cells stably expressing fluorescent protein-tagged STING (mNeonGreen-STING) and TBK1 (TBK1-mScarletI). We found that after STING stimulation, TBK1 became associated with the trans-Golgi network (TGN), not the other parts of the Golgi. STING variants that constitutively induce the type I interferon response have been identified in patients with autoinflammatory diseases named "STING-associated vasculopathy with onset in infancy (SAVI)". Even in cells expressing these constitutively active STING variants, TBK1 was found to be associated with TGN, not the other parts of the Golgi. These results suggest that TGN acts as a specific platform where STING associates with and activates TBK1.Key words: the Golgi, membrane traffic, innate immunity, STING.

Cyclic GMP-AMP （cGAMP） synthase （cGAS） and stimulator of interferon genes （STING） are the two key molecules that trigger type I interferon reponse upon the emergence of cytosolic “non-self DNA”，such as the one from DNA viruses. Recent studies have shown that this pathway is also activated with “self-DNA” leaked out from the nucleus or mitochondria. Moreover，the cGAS-STING pathway is revealed to be associated with various autoinflammatory，autoimmune，and neurodegenerative diseases. In this review，we particularly focus on emerging issues regarding the regulation of STING activity by membrane traffic. The dysregulated membrane traffic of STING are recently shown by us and others to underly the pathogenesis of autoinflammatory diseases，SAVI （STING-associated vasculopathy with onset in infancy） and COPA （a monogenic autoinflammatory disease caused by missense mutations of coatomer protein complex subunit α （α-COP））.

The Rab family of small GTPases comprises the largest number of proteins (∼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.

Rab7 (or Ypt7 in yeast) is one of the well-characterized members of the Rab family small GTPases, which serve as master regulators of membrane trafficking in eukaryotes. It localizes to late endosomes and lysosomes and has multiple functions in the autophagic pathway as well as in the endocytic pathway. Because Rab7/Ypt7 has previously been shown to regulate the autophagosome-lysosome fusion step in yeast and fruit flies (i.e., autophagosome accumulation has been observed in both Ypt7-knockout [KO] yeast and Rab7-knockdown fruit flies), it is widely assumed that Rab7 regulates the autophagosome-lysosome fusion step in mammals. A recent analysis of Rab7-KO mammalian cultured cells, however, has revealed that Rab7 is essential for autolysosome maturation (i.e., autolysosome accumulation has been observed in Rab7-KO cells), but not for autophagosome-lysosome fusion, under nutrient-rich conditions. Thus, although Rab7/Ypt7 itself is essential for the proper progression of autophagy in eukaryotes, the function of Rab7/Ypt7 in autophagy in yeast/fruit flies and mammals must be different. In this review article, we describe novel roles of Rab7 in mammalian autophagy and discuss its functional diversification during evolution.

Tunneling nanotubes (TNTs) are actin-enriched membranous channels enabling cells to communicate over long distances. TNT-like structures form between various cell types and mediate the exchange of different cargos, such as ions, vesicles, organelles and pathogens; thus, they may play a role in physiological conditions and diseases (e.g. cancer and infection). TNTs also allow the intercellular passage of protein aggregates related to neurodegenerative diseases, thus propagating protein misfolding. Understanding the mechanism of TNT formation is mandatory in order to reveal the mechanism of disease propagation and to uncover their physiological function. Vesicular transport controlled by the small GTPases Rab11a and Rab8a can promote the formation of different plasma membrane protrusions (filopodia, cilia and neurites). Here, we report that inhibiting membrane recycling reduces the number of TNT-connected cells and that overexpression of Rab11a and Rab8a increases the number of TNT-connected cells and the propagation of vesicles between cells in co-culture. We demonstrate that these two Rab GTPases act in a cascade in which Rab11a activation of Rab8a is independent of Rabin8. We also show that VAMP3 acts downstream of Rab8a to regulate TNT formation.

Macroautophagy (simply called autophagy hereafter) is an intracellular degradation mechanism that is activated by nutrient starvation. Although it is well known that starvation induces autophagosome formation in an mTORC1-dependent manner, whether starvation also regulates autophagosome or autolysosome maturation was unclear. In the present study, we succeeded in demonstrating that starvation activates autolysosome maturation in mammalian cells. We found that knockout (KO) of Rab7 (herein referring to the Rab7a isoform) caused an accumulation of a massive number of LC3-positive autolysosomes under nutrient-rich conditions, indicating that Rab7 is dispensable for autophagosome-lysosome fusion. Intriguingly, the autolysosomes that had accumulated in Rab7-KO cells matured and disappeared after starvation for a brief period (∼10 min), and we identified glutamine as an essential nutrient for autolysosome maturation. In contrast, forced inactivation of mTORC1 through treatment with its inhibitor Torin2 failed to induce autolysosome maturation, suggesting that the process is controlled by an mTORC1-independent mechanism. Since starvation-induced autolysosome maturation was also observed in wild-type cells, the nutrient-starvation-induced maturation of autolysosomes is likely to be a generalized mechanism in the same manner as starvation-induced autophagosome formation. Such multistep regulatory mechanisms would enable efficient autophagic flux during starvation.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.