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Protistology ■ 53
Two epigenetic phenomena occur in crosses of Paramecium tetraurelia strains 32 and 51. Strain 32 is deficient for an IES present in one of the mating type genes, mtB, of strain 51. Internal eliminated sequences are excised from the developing macro-nuclear genome by a fascinating mechanism of genomic subtraction mediated by scanRNAs. However, if an IES is present in genome of one partner but absent in genome of another, then F1 hybrids deriving from the latter parent are unable to excise such IES from developing somatic genome: they can't produce a certain scanRNA. Moreover, F2 progeny of such cell will inherit this IES retained in macronucleus. IES inside a gene disrupts its function, thus reminding hybrid dysgenesis known for Drosophila. Indeed, in 25% of crosses we observed loss of mtB function in F2 progeny derived from parent 32. We also found unexpectedly that in 20% of crosses IES in mtB gene was retained in macronucleus of F2 progeny derived from parent 51, which normally produces scanRNAs and excises this IES. Analogous phenomenon was reported in cross of d12 and d48 deletion mutants of P. tetraurelia restoring functional gene of surface antigen A. We suggest that its mechanism may be connected with hemizygocity state ofthe deleted locus in F1 hybrids of such crosses, leading somehow to deviation of such sequence excision despite scanRNAs for it are present. These epigenetic effects may contribute into speciation in ciliates, as occasional hemizygocity may lead to lethality of interstrain hybrids. Supported by RFBR 16-04-01710.
RECONSTRUCTION OF CELLULAR SHAPE DEFORMATION THROUGH CONTRACTION OF CORTEX ACTOMYOSIN Nishigami Yukinori1, Ito Hiroaki2, Sonobe Seiji3, Ichikawa Masatoshi1
1 - Department of Physics, Graduate School ofScience, Kyoto University, Kyoto 606-8502, Japan
2 - Department ofMechanical Engineering, Graduate School ofEngineering, Osaka University, Osaka 5650871, Japan
3 - Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan [email protected]
Giant free-living amoebae, Amoeba proteus, actively deform cellular shape during the locomotion. The deformation is induced by contraction of cortical actin and myosin (actomyosin). In the process, since actomyosin is connected to the cellar membrane and transmit the generated force to deform the membrane. Although the contractile properties of
actomyosin networks have been reported, actual contributions to the membrane deformation are still unclear because of the cellular complexities. Here, in order to simplify the complex system, we attempted to reconstitute a simple model system, in which lipid monolayer was deformed by actomyosin. In living cells, the connection between actomyosin and lipid layer is achieved by various types of proteins. To simply accomplish the actin-membrane connection in vitro, we adapted positively-charged lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), expecting the electrostatic adhesion between negatively-charged actin and DOTAP. We extracted actomyosin from A. proteus and enclosed actomyosin fraction within a spherical space surrounded by a DOTAP monolayer. As a result, active deformation ofthe lipid monolayer was yielded. From analyses ofthe static and dynamic properties of the deformation, we found that the depth and width ofthe deformation were dependent on the curvature radius of the sphere. The observed curvature dependence is explained by the theoretical description including elasticity and contractility of the cortex. Our results provide a fundamental insight into the cellular membrane deformation induced by the actomyosin cortex during amoeboid locomotion. For more details, see Nishigami et al. (Sci. Rep. 6, 19864, 2016) and Ito, Nishigami et al. (Phys. Rev. E92,062711,2015).
NUCLEAR DIVISION PROCESS IN TESTATE AMOEBA PAULINELLA CHROMATOPHORA Nomura M., Ishida K.
Faculty ofLife and Environmental Sciences, University of Tsukuba
Paulinella chromatophora is a euglyphid testate amoeba (Rhizaria, Cercozoa) living in a shell composed of ~50 rectangular siliceous scales. In this species, the complex shell construction process appears to be integrated under the cell cycle regulation, since the cell division does not proceed without the completion of shell construction. Before cell division, scales produced inside of mother cell are secreted out from the cell and assembled into a new shell by a specialized thick pseudopodium. Following the completion of shell construction, one of daughter cells moves into the new shell. Despite that knowledge, it is still unknown how the cell division process proceeds in response to the shell construction. In this study, we focused on how the nucleus divides along with shell construction process in P. chromatophora. In an intermediate stage of shell construction, the nucleus in the maternal cell was in prophase. In this phase, the nucleolus, which
54 • "PROTIST—2016
is prominent in interphase, was disappeared and chromosomes were scattered in the nucleus. The newly formed shell was almost or fully constructed when the nuclear division reaches metaphase. In this phase, the spindle body was formed and the chromosomes were arranged at the equatorial plane randomly. At the time of completion of shell construction, the nucleus was observed to be in anaphase, and chromosomes were separated into anterior and posterior side of the nucleus. After the migration of a daughter cell into new shell, the nucleus with densely condensed chromosomes was observed to locate at posterior end of each daughter cell.
MITOSOMES IN ENTAMOEBA HISTOLYTICA. DIFFERENTIATION, METABOLITE TRANSPORT, AND FISSION Nozaki T.12
1 - National Institute of Infectious Diseases
2 - University of Tsukuba [email protected]
Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) in anaerobic/ microaerophilic eukaryotes with highly reduced and divergent functions. Entamoeba possesses a highly divergent MRO known as the mitosome. The biological functions and their origin of Entamoeba mitosomes have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. We recently purified and identified cholesteryl sulfate (CS) as a final sulfate activation metabolite. We further identified the gene encoding the cholesteryl sulfotransferase responsible for synthesis of CS. Supplementation of CS to the culture increased the number of cysts, while, conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate activation pathway, inhibited cyst formation. These results indicate that CS plays an important role in differentiation, an essential process for transmission of Entamoeba between hosts. Furthermore, Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, also has the sulfate activation pathway in MROs, but does not possess the capacity for CS production. Hence, we proposed that a unique function of MROs in Entamoeba contributes to adaptation of its parasitic life cycle. Understanding of metabolite trafficking across the two mitosomal membranes is important to understand metabolic functions ofmitosomes. We recently discovered a novel mitosomal P-barrel outer membrane protein of30 kDa (MBOMP30) and several novel membrane-spanning
proteins from a list of the mitosome proteome. We experimentally confirmed their localization and integration to mitosome membranes by Percoll-gradient fractionation, carbonate fractionation, immunofluorescence assay, and immunoelectron microscopy. These new class ofmitosomal membrane proteins including MBOMP30 likely play unique and indispensable roles in Entamoeba mitosomes. We also found that two dynamin-related proteins, DrpA and DrpB, are involved in mitosome fission. Expression of a mutant form or gene silencing of these Drps caused abnormal morphology of mitoses and growth defect, suggesting that mitosome fission is mediated in part by these Drps.
MORPHOLOGY, PHYLOGENY, AND TRANS-CRIPTOME DATA OF A NEW ANAEROBIC METOPUS SPECIES (CILIOPHORA, ARMO-PHORIDA) FROM YANTAI, CHINA Omar A., Zhang Q., Gong J. Laboratory of Microbial Ecology and Matter Cycles, Yantai Institute of Coastal Zone Research, Chinese Academy ofScience, Yantai, China [email protected]
A new anaerobic Metopus species was discovered in a soil sample from fruit garden in Yantai, China, and investigated using morphological, morphometrical, and molecular methods. The morphology was studied using in vivo observation and protargol impregnation. The main features ofthe new Metopus species include: (i) size in vivo 75-105 * 35-55 ^m; (ii) body shape ellipsoidal to pyriform; (iii) nuclear apparatus invariably in preoral dome, macronucleus reniform, micronucleus globular to ellipsoidal attached to macronucleus; (iv) cytoplasm studied with lipid droplets especially in preoral dome; (v) five perizonal and 18-21 somatic ciliary rows of which three extend onto preoral dome (dome kineties); (vi) three to five distinctly elongated caudal cilia; and (vii) adoral zone composed of 21-29 membranelles and distinctly shorter than perizonal ciliary stripe (45% vs. 61% ofbody length on average). Moreover, this species contains numerous, conspicuous hydrogenosomes, anaerobically-functioning mito-chondrial-related organelles, as an adaptation for the anaerobic lifestyle. SSU rRNA and mRNA were obtained using a single cell transcriptome protocol, and were sequenced with both Sanger and MiSeq Illumina technology. The obtained data were used for phylogenomic analyses and analyzing basic metabolic processes of this anerobic ciliate, including searching for genes ofputative anaerobic-adapting functions.
Supported by the NSFC project 31550110213 and the CAS project 2015PB040.
