Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions

E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.     

Polychaeta of the Kara and Pechora seas: Data of the 2012 trawl survey

The species composition of the polychaetes derived from ichthyological and Sigsbee trawls in the Pechora and Kara seas in 2012 was studied and compared with the grab survey data of 1993–1995. The distribution of the large sabellidae, nektobenthic, and bathypelagic species that are poorly caught by a grab has been determined for the first time. Changes that were observed in the biogeographical polychaete structure in the Kara Sea (a higher proportion of the boreal species and a lower proportion of the Arctic species) may reflect a response of zoobenthos to the Arctic warming in the late 20th and early 21st centuries.     

Anthozoa from the northern Mid-Atlantic Ridge and Charlie-Gibbs Fracture Zone

An annotated list of deep-sea Anthozoa of the orders Actiniaria, Antipatharia, Scleractinia, Alcyonacea and Pennatulacea collected on the G.O. Sars MAR-ECO cruise to the Mid-Atlantic Ridge between the Azores and the southern tip of the Reykjanes Ridge is given. A total of 33 species is reported of which 32 were identified to species or genus level. The groups most rich in species were Actiniaria (nine species), Scleractinia (eight species) and Pennatulacea (eight species). Scleractinia, Antipatharia and Pennatulacea were mainly represented by species with a wide or cosmopolitan geographical distribution. In contrast, most of the actiniarians had been rarely recorded in the North Atlantic. Three species, Schizopathes affinis Brook, 1889 (Antipatharia), Dendrobrachia multispina Opresko & Bayer, 1991 and Heteropolypus cf. insolitus Tixier-Durivault, 1964 (Alcyonacea) are reported from the North Atlantic for the first time.     

Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F₁ sector catalyzes ATP synthesis, whereas the F_o sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F₁ and F_o sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F_o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F_o sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.     

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro ^R, copGFP, or H-2K ^k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.