NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.     

Animal models for respiratory chain disease

Elucidation of the pathogenesis in respiratory chain diseases is of great importance for developing specific treatments. The limitations inherent to the use of patient material make studies of human tissues often difficult and the mouse has therefore emerged as a suitable model organism for studies of respiratory chain diseases. In this review, we present an overview of the field and discuss in depth a few examples of animal models reproducing pathology of human disease with primary and secondary respiratory chain involvement.     

Social status and reproductive success of male Macaca fascicularis

Dominant males of nonseasonally breeding species should have more opportunity than seasonal breeders to monopolize access to estrous females and thus enhance their reproductive success. They may also use other strategies for maximizing reproductive success such as producing offspring by relatively high ranking females. To test these hypotheses, the paternity of 35 (80%) of 44 Macaca fascicularis offspring, born over a 28-month period, was established using electrophoretically and serologically defined genetic markers. The social ranks of each of the seven potential fathers and 26 potential mothers were determined by recording outcomes of dyadio agonistic interactions. No evidence could be found to support either hypothesis. Further, in view of previously reported significant, positive correlations between male social rank and sexual activity rates in this study group, sexual activity rates do not appear to predict the number of offspring a male sires in this species.     

A new species of Galearis (Orchidinae: Orchidaceae) from Sichuan,China

A new species, Galearis huanglongensis Q.W.Meng & Y.B.Luo, is described and illustrated. It is similar to Galearis cyclochila (Franch. & Sav.) Soó and Galearis diantha (Schltr.) P.F.Hunt, but differs in having a short spur, two elliptical lateral stigma lobes and distinctly separated bursicles. This new species is known only from the type locality, the Huanglong Valley, Songpan County, western Sichuan, China, growing amongst mosses under alpine shrubs at an elevation of about 3000 m. Based on two years of observations of its population size, the species was categorized as critically endangered CR (B1a, B2a) according to the World Conservation Union (IUCN) Red List Categories and Criteria, Version 3.1. The micromorphology of pollinia and seeds was observed by scanning electron microscopy and compared with that of G. cyclochila and G. diantha. The results supported G. huanglongensis Q.W.Meng & Y.B.Luo as a new species. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 689–695.     

Genetic markers for identification of Patagonian toothfish and Antarctic toothfish

Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Muscle fine structure reflects ecotype in two nototheniids

The fine structure of swimming (pectoral) and myotomal (axial) skeletal muscle and myocardium of two species of Antarctic nototheniid fishes were studied by electron microscopy, comparing the cryopelagic Pagothenia borchgrevinki and the benthic Trematomus bernacchii . Mean fibre size varied by a factor of four among muscles within each species and may have reflected the locomotory power available, being larger in pectoral oxidative (red) and axial glycolytic (white) muscle of P. borchgrevinki . Both species use labriform locomotion, and the more active P. borchgrevinki had a greater capillary supply, expressed as a capillary to fibre ratio, than T. bernacchii to both red (3·48 ± 0·36 v . 1·63 ± 0·14, mean ±  s . e .; P  < 0·01) and white (2·70 ± 0·20 v . 1·53 ± 0·18, mean ±  s . e .; P  < 0·01) regions of the pectoral musculature. The greater aerobic scope of P. borchgrevinki was strikingly demonstrated in the higher mitochondrial content of all skeletal muscle types sampled, and the ventricular myocardium (0·269 ± 0·011 v . 0·255 ± 0·012 mean ±  s . e .; P  < 0·05). Minor differences were found in other elements of fibre composition, with the exception of a five‐fold greater lipid content in pectoral red fibres of P. borchgrevinki (0·074 ± 0·014 mean ±  s . e .) v . T. bernacchii (0·010 ± 0·003; P  < 0·05). Differences in muscle fine structure among species clearly reflected differences in their ecotype.     

Implicit associations with social status: the effects of relationship involvement

The study tested the proposition that relationship involvement influences the implicit responses of women to high- and low-status professions. It was hypothesized that when a high-involvement context was primed, women would have more positive implicit associations with high-status occupations than when a low-involvement context was primed. In contrast, when a high-involvement context was primed, women would have more negative associations with low-status occupations than when a low-involvement context was primed. To test the hypothesis, 123 female participants received a high or low relationship involvement prime. Then the participants completed a single category implicit associations test designed to measure the participants' associations with either high- or low-status occupations. As predicted, the relationship involvement prime influenced the positivity of associations made with high- and low-status occupations. The study pointed to the possibility that persons possess evolutionary-based implicit associations.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Diversity measures in macroinvertebrate and zooplankton communities related to the trophic status of subtropical reservoirs: Contradictory or complementary responses?

Macroinvertebrate communities have been widely used as a tool for assessing the environmental quality of freshwater ecosystems, whereas zooplankton communities have been to some extent neglected. However, the importance of using different indicators to achieve a more comprehensive framework of assessment regarding water quality has been recognized. This study compared estimates of species richness (number of species) and the Shannon–Wiener index for data on macroinvertebrate and zooplankton communities in tropical reservoirs and related them to their trophic state. The trop+hic classification was obtained by applying the Carlson index (1977) modified by Toledo et al. (1983), and the index of the Brazilian Society of the Environmental Technology Agency. The comparative response of the different indicators was analyzed using a series of bivariate correlations (Draftsman’s plot). The results illustrate that diversity measures, namely species richness, responded differently when related to the trophic classification of reservoirs, depending on the community considered. The species richness of zooplankton was positively related to hypereutrophic conditions, due to the higher number of rotifer species, including tolerant generalist species and at the same time, as a result of the exclusion of species from other groups, whereas for macroinvertebrates, species richness was negatively related to hypereutrophic conditions. Melanoides tuberculatus, which exhibits a high tolerance and competitive ability under such conditions, was the dominant species in macroinvertebrate communities, which excluded endemic species and reduced local richness and diversity. The same indicators applied to the zooplankton and macroinvertebrate communities might therefore provide contradictory responses regarding ecological quality assessment in tropical reservoirs, which suggest that zooplankton should be taken into account among the biological quality elements considered in the ecological quality assessment, management, and restoration of water bodies.