Massimo Micaroni

Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid cycle (TCA cycle) that catalyses the hydration of fumarate into malate. Germline mutations of FH are responsible for hereditary leiomyomatosis and renal-cell cancer (HLRCC). It has previously been demonstrated that the absence of FH leads to the accumulation of fumarate, which activates hypoxia-inducible factors (HIFs) at normal oxygen tensions. However, so far no mechanism that explains the ability of cells to survive without a functional TCA cycle has been provided. Here we use newly characterized genetically modified kidney mouse cells in which Fh1 has been deleted, and apply a newly developed computer model of the metabolism of these cells to predict and experimentally validate a linear metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion from Fh1-deficient cells. This pathway, which involves the biosynthesis and degradation of haem, enables Fh1-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. We predicted and confirmed that targeting this pathway would render Fh1-deficient cells non-viable, while sparing wild-type Fh1-containing cells. This work goes beyond identifying a metabolic pathway that is induced in Fh1-deficient cells to demonstrate that inhibition of haem oxygenation is synthetically lethal when combined with Fh1 deficiency, providing a new potential target for treating HLRCC patients.

Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin

Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. Here we demonstrate that Drosophila Atlastin—the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia—localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion.

Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures.

Bi-directional calcium (Ca(2+)) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca(2+) stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca(2+) release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca(2+) stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR-mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.

Src kinase regulates the integrity and function of the Golgi apparatus via activation of dynamin 2

The size and integrity of the Golgi apparatus is maintained via a tightly controlled regulation of membrane traffic using a variety of different signaling and cytoskeletal proteins. We have recently observed that activation of c-Src has profound effects on Golgi structure, leading to dramatically vesiculated cisternae in a variety of cell types. As the large GTPase dynamin (Dyn2) has been implicated in Golgi vesiculation during secretion, we tested whether inhibiting Dyn2 activity by expression of a Dyn2K44A mutant or siRNA knockdown could attenuate active Src-induced Golgi fragmentation. Indeed, these perturbations attenuated fragmentation, and expression of a Dyn2Y(231/597)F mutant protein that cannot be phosphorylated by Src kinase had a similar effect . Finally, we find that Dyn2 is markedly phosphorylated during the transit of VSV-G protein through the TGN whereas expression of the Dyn2Y(231/597)F mutant significantly reduces exit of the nascent protein from this compartment. These findings demonstrate that activation of Dyn2 by Src kinase regulates Golgi integrity and vesiculation during the secretory process.

Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca2+ release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1Y522S/WT), the mutation causes SR Ca2+ leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1Y522S/WT knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1Y522S/WT mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2–4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1Y522S/WT mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca2+ sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

Electron Tomography Reveals Rab6 Is Essential to the Trafficking of trans-Golgi Clathrin and COPI-Coated Vesicles and the Maintenance of Golgi Cisternal Number

We have shown previously that Rab6, a small, trans‐Golgi‐localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high‐resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans‐Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin‐coated vesicles and the smaller to coat protein I (COPI)‐coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans‐Golgi/trans‐Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6‐depleted HeLa cells that we observed may well be coupled to the trans‐Golgi accumulation of COPI‐coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6‐dependent protein machine affecting Golgi‐proximal, coated vesicle accumulation and probably transport at the trans‐Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

PARP16/ARTD15 Is a Novel Endoplasmic-Reticulum-Associated Mono-ADP-Ribosyltransferase That Interacts with, and Modifies Karyopherin-ß1

Background Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose) polymerase (PARP/ARTD) family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain. Methodology/Principal Findings Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme. Conclusions/Significance Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.

Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: Novel clues on the route to bacterial biogenesis of selenium nanoparticles

A putative biosynthetic mechanism for selenium nanoparticles (SeNPs) and efficient reduction of selenite (SeO32-) in the bacterial strain Stenotrophomonas maltophilia SeITE02 are addressed here on the basis of information gained by a combined approach relying on a set of physiological, chemical/biochemical, microscopy, and proteomic analyses. S. maltophilia SeITE02 is demonstrated to efficiently transform selenite into elemental selenium (Se°) by reducing 100% of 0.5mM of this toxic oxyanion to Se° nanoparticles within 48h growth, in liquid medium. Since the selenite reducing activity was detected in the cytoplasmic protein fraction, while biogenic SeNPs showed mainly extracellular localization, a releasing mechanism of SeNPs from the intracellular environment is hypothesized. SeNPs appeared spherical in shape and with size ranging from 160nm to 250nm, depending on the age of the cultures. Proteomic analysis carried out on the cytoplasmic fraction identified an alcohol dehydrogenase homolog, conceivably correlated with the biogenesis of SeNPs. Finally, by Fourier Transformed Infrared Spectrometry, protein and lipid residues were detected on the surface of biogenic SeNPs. Eventually, this strain might be efficaciously exploited for the remediation of selenite-contaminated environmental matrices due to its high SeO32- reducing efficiency. Biogenic SeNPs may also be considered for technological applications in different fields.

Axenic Culture of a Candidate Division TM7 Bacterium from the Human Oral Cavity and Biofilm Interactions with Other Oral Bacteria

ABSTRACT The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 μm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.