NLRP3 activation in response to disrupted endocytic traffic

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3.

Nitric oxide may regulate focal adhesion turnover and cell migration in MDA-MB-231 breast cancer cells by modulating early endosome trafficking

Cell migration is an essential process for wound healing, metastasis and inflammation. Focal adhesions (FA) are local regions of plasma membrane consisting of multiprotein complexes providing adhesive contact between the cell and the extracellular matrix (ECM). FA turnover regulates different signalling pathways implicated in various cellular responses (e.g. cell migration). Endocytosis, specifically the dynamin and clathrin pathways, is known to regulate cell migration by modulating FA dynamics. In this study, we investigated whether NO activity regulates cell migration, FA dynamics and early endosome trafficking in MDA-MB-231 cells. The assessment of cell migration showed a slowing down of cell migration and an increased duration of FA turnover in cells treated with inhibitors of NO synthase (NOS) such as L-NAME or 1400W. In addition, these treatments were found to exhibit no effect on transferrin and dextran uptake mediated by endocytosis and micropinocytosis, respectively. The number of early endosome antigen 1 (EEA1)-positive endosomes was reduced while their sizes were found to increase in cells treated with L-NAME or 1400W. In contrast, these inhibitors did not affect the number nor the size of Rab5-positive endosomes. Furthermore, we demonstrated that EEA1, endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) were colocalised. Using the biotin switch assay followed by western blot, we showed that early endosome proteins such as APPL1, EEA1, Rab5 were found to be S-nitrosylated. These results were further supported by the sequence analysis performed with the GPS-SNO algorithm which predicted the S-nitrosylation of these endosomal proteins. Taken together, our findings suggest that NO might be involved in cell migration and FA turnover through early endosome trafficking in MDA-MB-231 cells. Running title: Nitric oxide in MDA-MB-231 breast cancer cells

The Alzheimer's disease genetic risk factor BIN1 induces isoform-dependent neurotoxicity through early endosome defects

The Bridging Integrator 1 (BIN1) gene is a major genetic risk factor for Alzheimer's disease (AD) but little is known about its physiological functions. In addition, deciphering its potential pathophysiological role is difficult due to its numerous isoforms expressed in different cerebral cell types. Here we took advantage of a drosophila model to assess in vivo the impact of different BIN1 isoforms on neuronal toxicity: the neuronal isoform 1 (BIN1iso1), the muscular isoform 8 (BIN1iso8) and the ubiquituous isoform 9 (BIN1iso9). We showed that contrary to BIN1iso8 and BIN1iso9, BIN1iso1 overexpression induced neurodegeneration and an accumulation of vesicles mainly labeled by endosome markers. Systematic search for endosome trafficking regulators that are able to rescue BIN1iso1-induced neurodegeneration indicated a defect in the early endosome trafficking machinery. In human induced neurons and cerebral organoids, BIN1 knock-out resulted in narrowing of the early endosomes. This phenotype was rescued by BIN1iso1 expression but not that of BIN1iso9. Finally, in accordance with our previous observation in flies, we also observed that BIN1iso1 overexpression led to an increase in size of the early endosomes in human induced neurons. Altogether, our data demonstrate that the AD genetic risk factor BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation which is a very early pathophysiological feature observed in AD pathogenesis.

Dynamic Dissection of the Endocytosis of Porcine Epidemic Diarrhea Coronavirus Cooperatively Mediated by Clathrin and Caveolae as Visualized by Single-Virus Tracking

ABSTRACT Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection. IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.

ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. Here, we demonstrate that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). We further demonstrate that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.

Lipid Transfer–Dependent Endosome Maturation Mediated by Protrudin and PDZD8 in Neurons

Endosome maturation refers to the conversion of early endosomes (EEs) to late endosomes (LEs) for subsequent fusion with lysosomes. It is an incremental process that involves a combination of endosome fusion and fission and which occurs at contact sites between endosomes and the endoplasmic reticulum (ER), with knowledge of the underlying mechanisms having increased greatly in recent years. Protrudin is an ER-resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking, whereas PDZD8 is a mammalian paralog of a subunit of the yeast ERMES (ER-mitochondrial encounter structure) complex that possesses lipid transfer activity. A complex of protrudin and PDZD8 was recently found to promote endosome maturation by mediating lipid transfer at ER-endosome membrane contact sites. This review focuses on the roles of the protrudin-PDZD8 complex in tethering of endosomes to the ER, in mediating lipid transfer at such contact sites, and in regulating endosome dynamics, especially in neuronal cells. It also addresses the physiological contribution of endosome maturation mediated by this complex to neuronal polarity and integrity.

Endocytic Pathway of Feline Coronavirus for Cell Entry: Differences in Serotype-Dependent Viral Entry Pathway

Feline coronavirus (FCoV) is a pathogen causing a lethal infectious disease in cats, feline infectious peritonitis. It has two serotypes (type I FCoV and type II FCoV). According to our previous study, type I FCoV infection is inhibited by compounds inducing intracellular cholesterol accumulation, whereas type II FCoV infection is not inhibited. Intracellular cholesterol accumulation was reported to disrupt late endosome function. Based on these findings, types I and II FCoV are considered to enter the cytosol through late and early endosomes, respectively. We investigated whether the antiviral activities of a late endosome trafficking inhibitor and cholesterol-accumulating agents are different between the FCoV serotypes. The late endosome trafficking inhibitor did not inhibit type II FCoV infection, but it inhibited type I FCoV infection. Type I FCoV infection was inhibited by cholesterol-accumulating triazoles, but not by non-cholesterol-accumulating triazoles. These phenomena were observed in both feline cell lines and feline primary macrophages. This study provides additional information on the differences in intracellular reproductive cycle between type I and type II FCoV.

PAK Kinases Target Sortilin and Modulate Its Sorting

ABSTRACT The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.