SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis.

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8+ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8+ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8+ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter-linked and important for CD8+ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL-2 production - as well as subsequent IL-2 dependent TNF, IFN-γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8+ T cells.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus.

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions.

Effector memory (EM) CD4(+) T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4(+) T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-γ. EM CD4(+) T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4(+) T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (ΔΨm) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved ΔΨm and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on ΔΨm, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates ΔΨm and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4(+) T cells in normoxic and hypoxic conditions.

Structural characterization of altered nucleoporin Nup153 expression in human cells by thin-section electron microscopy.

Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins Nup153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of Nup153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by Nup153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of Nup153. Moreover, even mild over-expression of Nup153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of Nup153 that is sufficient to induce these rearrangements. Our data indicate a central function of Nup153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior.

The small GTPase Arf1 modulates mitochondrial morphology and function.

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.

The ArfGEF GBF-1 Is Required for ER Structure, Secretion and Endocytic Transport in C. elegans.

Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic.

A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.

Notch signaling plays a fundamental role in cellular differentiation and has been linked to human diseases, including cancer. We report the use of comprehensive RNAi analyses to dissect Notch regulation and its connections to cellular pathways. A cell-based RNAi screen identified 900 candidate Notch regulators on a genome-wide scale. The subsequent use of a library of transgenic Drosophila expressing RNAi constructs enabled large-scale in vivo validation and confirmed 333 of 501 tested genes as Notch regulators. Mapping the phenotypic attributes of our data on an interaction network identified another 68 relevant genes and revealed several modules of unexpected Notch regulatory activity. In particular, we note an intriguing relationship to pyruvate metabolism, which may be relevant to cancer. Our study reveals a hitherto unappreciated diversity of tissue-specific modulators impinging on Notch and opens new avenues for studying Notch regulation and function in development and disease.

The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii.

Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.

The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

Caenorhabditis elegans teneurin, ten-1, is required for gonadal and pharyngeal basement membrane integrity and acts redundantly with integrin ina-1 and dystroglycan dgn-1.

The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells.

Biofilm formation induces C3a release and protects Staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing.

BACKGROUND: Biofilm formation is considered to be an important virulence factor of the opportunistic pathogen Staphylococcus epidermidis. We hypothesized that biofilm formation could interfere with the deposition of immunoglobulins and complement on the bacterial surface, leading to diminished activation of the complement system and protection from killing by human phagocytes. METHODS: The killing of biofilm-encased and planktonically grown wild-type (wt) S. epidermidis and the killing of an isogenic biofilm-negative ica mutant (ica(-)) by human polymorphonuclear neutrophils (PMNs) were compared. C3a induction and deposition of C3b and immunoglobulin G (IgG) on the bacteria after opsonization with human serum were assessed by enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. The virulence of the bacterial strains was compared in a mouse model of catheter-associated infection. RESULTS: Biofilm-embedded wt S. epidermidis was killed less well by human PMNs and induced more C3a than planktonically grown wt and ica(-) S. epidermidis. However, the deposition of C3b and IgG on the bacterial surface was diminished in biofilm-encased staphylococci. wt S. epidermidis was more virulent in implant-associated infections and was killed more slowly than ica(-) in ex vivo assays of killing by PMNs. CONCLUSIONS: The results indicate that prevention of C3b and IgG deposition on the bacterial surface contributes to the biofilm-mediated protection of S. epidermidis from killing by PMNs.

Domain topology of the p62 complex within the 3-D architecture of the nuclear pore complex.

The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex, which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. Here we show the localization of distinct domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C and N-terminal domains of p62 by immunogold-labeling using domain-specific antibodies against p62. In addition we expressed epitope-tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be of mobile and flexible nature.

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex.

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope.

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

Isolation of Bartonella schoenbuchensis from Lipoptena cervi, a blood-sucking arthropod causing deer ked dermatitis.

Bartonella schoenbuchensis, which commonly causes bacteremia in ruminants, was isolated from the deer ked Lipoptena cervi and was shown to localize to the midgut of this blood-sucking arthropod, causing deer ked dermatitis in humans. The role of B. schoenbuchensis in the etiology of deer ked dermatitis should be further investigated.

Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs.

Calcium-dependent protein kinases play a pivotal role in calcium signalling in plants and some protozoa, including the malaria parasites. They are found in various subcellular locations, suggesting an involvement in multiple signal transduction pathways. Recently, Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) has been found in the membrane and organelle fraction of the parasite. The kinase contains three motifs for membrane binding at its N-terminus, a consensus sequence for myristoylation, a putative palmitoylation site and a basic motif. Endogenous PfCDPK1 and the in vitro translated kinase were both shown to be myristoylated. The supposed membrane attachment function of the basic cluster was experimentally verified and shown to participate together with N-myristoylation in membrane anchoring of the kinase. Using immunogold electron microscopy, the protein was detected in the parasitophorous vacuole and the tubovesicular system of the parasite. Mutagenesis of the predicted acylated residues and the basic motif confirmed that dual acylation and the basic cluster are required for correct targeting of Aequorea victoria green fluorescent protein to the parasitophorous vacuole, suggesting that PfCDPK1 as the leishmanial hydrophilic acylated surface protein B is a representative of a novel class of proteins whose export is dependent on a 'non-classical' pathway involving N-myristoylation/palmitoylation.

The S. cerevisiae HtrA-like protein Nma111p is a nuclear serine protease that mediates yeast apoptosis.

The yeast S. cerevisiae can undergo programmed cell death that exhibits the typical cellular markers of apoptosis. The mammalian HtrA2 protein was recently reported to mediate apoptosis in a serine-protease-dependent manner owing to its ability to antagonise the inhibitor of apoptosis protein XIAP. Here, we report the identification and characterisation of the S. cerevisiae HtrA-like protein, which we termed Nma111p (for nuclear mediator of apoptosis), as a mediator of yeast apoptosis. Nma111p is a nuclear protein that, under cellular stress conditions (i.e. at elevated temperature or after induction of apoptosis by H2O2), tends to aggregate inside the nucleus without its expression level being upregulated, suggesting that aggregation of Nma111p is correlated to its death-mediating character. Nma111p belongs to the HtrA family of serine proteases and its pro-apoptotic activity depends on its serine-protease activity. Yeast cells that lack Nma111p survive better at 50 degrees C than wild-type cells and the cells show no apoptotic hallmarks, such as chromatin condensation and fragmentation, or accumulation of reactive oxygen species, after the induction of apoptosis by H2O2. By contrast, overexpression of Nma111p enhances apoptotic-like cell death. Therefore, Nma111p, like its mammalian homologue HtrA2, mediates apoptosis.