Identification of a new eukaryotic phylum ‘Picozoa’ in the oceanic environment

Recent molecular phylogenetic analyses of picoeukaryotic (< 3 µm) 18S rDNA environmental sequences revealed a deep branching lineage formally described as ‘picobiliphytes’ with unknown affinity to other eukaryotes. Until now, no cultured representatives existed to enable further investigation regarding the biodiversity and morphology of this newly erected clade. This work, reports on a newly-discovered, free-living eukaryotic protist Picomonas judraskeda gen. et sp. nov., from European marine coastal habitats which has a ‘picobiliphyte’ 18S rDNA signature. Its morphological and ultra-structural characters clearly show that it contains, neither chlorophyll nor phycobilin autofluorescence, both of which are main attributes of ‘picobiliphytes’. The phycobilin was assumed to have been derived from a secondary endosymbiont; and the host of the ‘picobiliphytes’ was postulated to be a sister to cryptophytes/ katablepharids. The isolate Picomonas is slightly elongated and 2.5-5 µm in length with two unequal flagella. It exhibits unique cell movements (jump, drag, and skedaddle mode of locomotion). Light and electron microscopic studies reveal that the cells are naked, the flagella not covered by hairs or scales and that a plastid is lacking. The cells thus are heterotrophic, although their food source could not be determined and food vacuoles containing bacteria are never observed. The cells harbor several unique compartments that do not match those of any other known eukaryotes. This uniqueness is corroborated by phylogenetic analyses of the complete nuclear ribosomal operon placed them into a new phylum ‘Picozoa’

Application of m- polar soft fuzzy bi- partite graph in residence selection process

An $m-$polar fuzzy set and soft sets are two different tools for representing uncertainty and vagueness. An $m-$polar soft fuzzy set is a mapping from parameter set to the $m-$polar fuzzy subsets of the universe. An $m-$polar soft fuzzy set theory provides a parameterized point of view for uncertainty modeling and soft computing model. In this paper, we have introduced the notions of $m-$polar soft fuzzy bipartite graph, size and degree of $m-$polar soft fuzzy bi-partite graph as well as an investigation on buying of residence by considering various parameters. People, while buying residence, have many options. So, to choose the best one, they have to consider many parameters. $m-$polar soft fuzzy graph is one of the major areas of graph theory, which finds a solution to this proble

<i>Picomonas judraskeda</i> Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes, Formerly Known as ‘Picobiliphytes’

<div><p>In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe <i>Picomonas judraskeda</i> gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for <i>Picomonas judraskeda</i>, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’.</p> </div

Flagellar root system in <i>Picomonas judraskeda</i>.

<p>7A. Non-consecutive serial sections from dorsal to ventral (sections are oblique to cell’s right). Each basal body is connected to two microtubular flagellar roots. The anterior root 1 (Ar1) runs anteriorly to cell’s left. The Ar2 runs posteriorly, at the right side of the cell and passes the cleft (cl). The other two flagellar roots originate from the posterior basal body and extend towards the posterior part of the cell. One of the posterior flagellar roots (Pr1) runs on the left side of the cell. The other broader posterior flagellar root (Pr2), runs between the Ar1 and Pr1. 7B. The Pr2 with 6 microtubules (arrowheads) obliquely sectioned. 7C. A cell with the Pr2 passing the vacuolar cisterna and mitochondrion. 7D. Consecutive serial cross sections through the Pr2 located in a depression of the mitochondrion. AF/PF (anterior−/posterior flagellum); Ab/Pb (anterior−/posterior basal body); Ar1/Ar2 (Anterior microtubular flagellar roots 1 and 2); Pr1/Pr2 (posterior microtubular flagellar roots 1 and 2); G (Golgi body); M (mitochondrion); vc (vacuolar cisterna); P (posterior digestive body); CMT (secondary cytoplasmic microtubule). Numbers at the top right indicate the number of the serial section. Scale bar: 200 nm.</p

Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome

Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates

Flagellar apparatus.

<p>5A. Longitidinal section of <i>Picomonas</i> from ventral to dorsal (sections are oblique from anterior part). Anterior flagellum (AF) and posterior flagellum (PF). The anterior flagellum locates close to the ventral surface followed more interiorly by the posterior flagellum. The basal bodies of both flagella are connected by a proximal connecting fiber (pcf) and exhibit two transitional regions (the proximal is termed ‘tr1’ and the distal ‘tr2’). 5B, C. Consecutive serial cross sections and two serial longitudinal sections of the anterior flagellum. Serial sections of 5B correspond to 5C, denoted in 5B_a-j at the left lower end. a. The axoneme with 9outer doublet and 2 central pair microtubules. b. The distal trasitional region (tr2) is involved in flagellar shedding (* indicates electron dense material near outer doublets). f. the central pair of microtubules orginate. h. the transitional region 1 (tr1), C-tubules added (arrowhead indicates a microtubulular triplet and arrow indicates a microtubular doublet. i,j. cross sections through basal body with microtubular triplets arranged in the clockwise direction, the basal body lumen is filled with electron dense material. AF/PF (anterior−/posterior flagellum); tr₁, tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); Ab/Pb (anterior−/posterior basal body); pcf (proximal connecting fiber); G (Golgi body); N (nucleus); Pr2 (posterior microtubular flagellar root 2). Numbers at the top right indicate the serial section. Scale bar: 100 nm.</p

Basal apparatus and probasal bodies.

<p>6A. Electron micrograph shows consecutive serial sections of <i>Picomonas judraskeda</i> from left to right (Sections are angled about 30 degree towards left from central axis). Each probasal body diverges from its parental basal body at right angles, both probasal bodies are oriented towards the ventral surface of the cell and are attached to the plasma membrane. 6B. 3D- scheme of the arrangement of basal bodies with probasal bodies. 6C. A schematic presentation of the flagellar basal apparatus seen from the ventral surface of the cell with proximal parts of the axonemes, basal bodies and four microtubular flagellar roots (for details see text). Numbers in brackets indicate the number of microtubules in each root. AF/PF (anterior−/posterior flagellum); tr₁,tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); Ab/Pb (anterior−/posterior basal body); Apb/Ppb (anterior/posterior probasal body); pcf (proximal connecting fiber); Ar1/Ar2 (Anterior microtubular flagellar roots 1 and 2); Pr1/Pr2 (posterior microtubular flagellar roots 1 and 2); G (Golgi body); N (nucleus); M (mitochondrion); cy (cytostome); P (posterior digestive body). Numbers at the top right indicate the number of the serial section. Scale bar: 200 nm.</p

Unrooted randomized accelerated maximum likelihood (RAxML) phylogenetic tree of partial nuclear SSU rDNA of Picozoa.

<p>The phylogeny is based on 1253 aligned characters of the SSU rDNA and includes 201 sequences of Picozoa. Most sequences are database entries derived from clone libraries (nine environmental sequences generated from a sample taken at Helgoland Roads and one sequence from <i>Picomonas judraskeda</i> are new sequences; accession numbers of the newly determined sequences are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059565#pone.0059565.s002" target="_blank">Fig. S2</a>). Bootstrap values >60% and posterior probabilities >0.80 for the three methods of analyses used (RAxML/NJ by PAUP/MrBayes) are shown on the respective branches. Branches in bold show maximal (100%/1.00) support. Labeling of clades (‘BP1–3′) followed [Cuvelier et al. 2008], 12 novel clades (‘P1–P13’) are recognized (for details see Results). The sequence of <i>Picomonas judraskeda</i> (in bold) was positioned in clade ‘P3’ (shaded).</p

A <i>Picomonas</i> cell.

<p>2A. Differential interference contrast of a chemically fixed cell. Inset shows phase contrast image of a live cell from tissue culture flask photographed with an inverted microscope (Scale bar 5 µm). 2B. Fluorescence and phase contrast overlay, nucleus (blue), mitochondrion (red). 2C. SEM image. 2D. A longitudinal section through a cell in the plane of the flagella, viewed from the cell’s left. 2E. A 3 D serial section reconstruction of the cell depicted in 2D. AF/PF (anterior−/posterior flagellum); AP/PP (anterior/posterior part of the cell); G (Golgi body); M (mitochondrion); MB (‘microbody’); N (nucleus); tr₁,tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); P (posterior digestive body); Cl (cleft separating the anterior from the posterior part of the cell); vc (vacuolar cisterna).</p

Feeding apparatus.

<p>4A. Longitudinal sections of a <i>Picomonas</i> cell from ventral to dorsal with the feeding apparatus in cross section. Sections 2 to section 7 depict a recently formed food vacuole inside the ‘basket’ of the feeding apparatus. 4B & C. Cross sections through two cells of <i>Picomonas</i> from anterior to posterior; sections begin in the central part of the cell, posterior to the basal bodies. 4B. Cell, in the non-feeding mode, without a food vacuole within the basket. 4C. Cell during active feeding with a large food vacuole within the basket (black triangles); the food vacuole contains irregularly-shaped, ‘fuzzy’ material, presumably taken up by endocytosis through the cytostome (white triangles). Two rows of fibers representing the left (LF) and right (RF) margins of the basket accompany the food vacuole. 4D. An SEM image of <i>Picomonas</i> visualizing the left side of the cell. Note that the posterior flagellum has been shed at the tr2; white triangles indicate the cytostome region of the feeding apparatus. 4E. Longitudinal section through the basket near the cytostome. It shows approx. 60 rows of fibers arranged in parallel (white arrows depict fibers in the right margin of the basket). 4F. 3D model of the feeding apparatus with rows of fibers in parallel arrangement (white arrows) forming a basket (arrangement of the cell as in 4D). Note that the fibers are interconnected by thin filaments. The basket is open towards the top and right (i.e. towards the anterior and dorsal direction of the cell respectively), while it is closed at the bottom (the posterior end of the cell). On the left side of the basket (representing the cell’s ventral surface) the fibers are attached to the plasma membrane thus forming the narrow, slit-like cytostome. AF/PF (anterior−/posterior flagellum); G (Golgi body); LF/RF (left and right row of fibers of the basket); M (mitochondrion); MB (microbody); N (nucleus); P (posterior digestive body); Ar2 (anterior microtubular flagellar root 2); Pr1 (posterior microtubular flagellar root 1); Pr2 (posterior microtubular flagellar root 2); FV, (food vacuole); vc (vacuolar cisterna); cy (cytostome). Numbers at the top right indicate the serial section. Scale bar: 200 nm; except Fig. 4A, section 2 (100 nm).</p