Investigation of yeast genes possibly involved in mtDNA stability using the nematode Caenorhabditis elegans

Screening of Caenorhabditis elegans genes possibly involved in the mitochondrial genome maintenance was performed using our previous validated method of RNAi combined with ethidium bromide. This was to knock down C. elegans genes homologous to yeast genes known to be involved in mtDNA stability but of unknown molecular function or to identify transient components that could play important role on the stability of mtDNA in a temporal and/or spatial manner. C. elegans homologs for 11 genes among 27 yeast genes for which deletion leads to a rho0 state were found, however, only 5 genes were present in the RNAi library. Out of these 5 genes, 1 gene (homolog of GEM1) gave a clear L3 arrest on RNAi and ethidium bromide indicating its involvement on mtDNA stability. Four other genes homologs of MTG2, YER087W, AVL9 and RRG3 did not lead to L3 arrest even though their deletion in Saccharomyces cerevisiae leads to rho0 state. Although MTG2 has been reported to be important in the function and structure on mtDNA stability in yeast, our results did not support those findings in C. elegans. The human homolog of this gene (MIRO1) can be considered as a candidate gene involved in mtDNA stability and sequenced in patients with mtDNA depletion diseases.Keywords: mtDNA, Caenorhabditis elegans, nucleoid, RNAi, candidate genes, homolog, MIRO

A yeast-based screening unravels potential therapeutic molecules for mitochondrial diseases associated with dominant ant1 mutations

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mito-chondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degen-erative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P . Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations

Dissection of the Carboxyl-Terminal Domain of the Proteasomal Subunit Rpn11 in Maintenance of Mitochondrial Structure and Function

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative -helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes

Photometry of cometary nuclei: Rotation rates, colours and a comparison with Kuiper Belt Objects

We present time-series data on Jupiter Family Comets (JFCs) 17P/Holmes, 47P/Ashbrook-Jackson and 137P/Shoemaker-Levy 2. In addition we also present results from `snap-shot' observations of comets 43P/Wolf-Harrington, 44P/Reinmuth 2, 103P/Hartley 2 and 104P/Kowal 2 taken during the same run. The comets were at heliocentric distances of between 3 and 7 AU at this time. We present measurements of size and activity levels for the snap-shot targets. The time-series data allow us to constrain rotation periods and shapes, and thus bulk densities. We also measure colour indices (V-R) and (R-I) and reliable radii for these comets. We compare all of our findings to date with similar results for other comets and Kuiper Belt Objects (KBOs). We find that the rotational properties of nuclei and KBOs are very similar, that there is evidence for a cut-off in bulk densities at ~ 0.6 g cm^{-3} in both populations, and the colours of the two populations show similar correlations. For JFCs there is no observational evidence for the optical colours being dependant on either position in the orbit or on orbital parameters.Comment: 15 pages, 19 figures, accepted for publication in MNRA

The nuclei of comets 7P/Pons-Winnecke, 14P/Wolf and 92P/Sanguin

Jupiter Family comets (JFCs) are short period comets which have recently entered the inner solar system, having previously orbited in the Kuiper Belt since the formation of the planets. We used two nights on the 3.6m New Technology Telescope (NTT) at the European Southern Observatory, to obtain VRI photometry of three JFCs; 7P/Pons-Winnecke, 14P/Wolf and 92P/Sanguin. These were observed to be stellar in appearance. We find mean effective radii of 2.24 \pm 0.02 km for 7P, 3.16 \pm 0.01 km for 14P and 2.08 \pm 0.01 km for 92P, assuming a geometric albedo of 0.04. From light-curves for each comet we find rotation periods of 7.53 \pm 0.10 and 6.22 \pm 0.05 hours for 14P and 92P respectively. 7P exhibits brightness variations which imply a rotation period of 6.8 \le P_rot \le 9.5 hours. Assuming the nuclei to be ellipsoidal the measured brightness variations imply minimum axial ratios a/b of 1.3 \pm 0.1 for 7P and 1.7 \pm 0.1 for both 14P and 92P. This in turn implies minimum densities of 0.23 \pm 0.08 g cm^{-3} for 7P, 0.32 \pm 0.02 g cm^{-3} for 14P and 0.49 \pm 0.06 g cm^{-3} for 92P. Finally, we measure colour indices of (V-R) = 0.40 \pm 0.05 and (R-I) = 0.41 \pm 0.06 for 7P/Pons-Winnecke, (V-R) = 0.57 \pm 0.07 and (R-I) = 0.51 \pm 0.06 for 14P/Wolf, and (V-R) = 0.54 \pm 0.04 and (R-I) = 0.54 \pm 0.04 for 92P/Sanguin.Comment: 12 pages, 14 figures, accepted for publication in A&

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders

The size distribution of Jupiter Family comet nuclei

We present an updated cumulative size distribution (CSD) for Jupiter Family comet (JFC) nuclei, including a rigourous assessment of the uncertainty on the slope of the CSD. The CSD is expressed as a power law, N(>r_N) \propto r_N^{-q}, where r_N is the radius of the nuclei and q is the slope. We include a large number of optical observations published by ourselves and others since the comprehensive review in the "Comets II" book (Lamy et al. 2004), and make use of an improved fitting method. We assess the uncertainty on the CSD due to all of the unknowns and uncertainties involved (photometric uncertainty, assumed phase function, albedo and shape of the nucleus) by means of Monte Carlo simulations. In order to do this we also briefly review the current measurements of these parameters for JFCs. Our final CSD has a slope q=1.92\pm 0.20 for nuclei with radius r_N \ge 1.25 km.Comment: 13 pages, accepted for publication in MNRA

Efficient Nuclear Transport of Structurally Disturbed Cargo: Mutations in a Cargo Protein Switch Its Cognate Karyopherin

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus