Phylogenetic tree of Ochrophyta using <i>rbc</i>L data to position <i>Guanchochroma wildpretii</i> and <i>Chrysopodocystis socialis</i>.

<p>Based on 74 photosynthetic taxa with 1231 positions. Maximum Likelihood subtree of Synchromophyceae and related photosynthetic organisms, with ML, MP and NJ bootstrap values and Bayesian posterior probabilities given at nodes. Formatting as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.g004" target="_blank">Fig 4</a>.</p

Characteristic features of Synchromophyceae and related amoeboid algae.

<p>* Under culture conditions multiple amoebae (>2) fusion was observed, forming large migrating/floating amoebae. It could not be determined whether these are cell aggregates (ectoplasmic fusion only) or fusion plasmodia (with endoplasmic fusion).</p><p>Features are taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.ref008" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.ref014" target="_blank">14</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131821#pone.0131821.ref068" target="_blank">68</a>] and this study. NAO—North Atlantic Ocean, MS—Mediterranean Sea, CS—Caribbean Sea, EC- English Channel.</p

Morphology of <i>Guanchochroma wildpretii</i>.

<p>(A) MCBs of young amoebae of <i>G</i>. <i>wildpretii</i> are encased in a thin, hyaline, globular lorica (arrowhead). Reticulopodia exit through the ostiole, which has a short rim (arrow), and fuse to form a meroplasmodium. (B) Migrating amoeba moving in the meroplasmodial network (white arrowhead). (C1–4) A migrating amoeba (arrow) moves on top of the meroplasmodium and settles close to it. Sequence of events from top left to bottom right (10:37 PM, 10:50 PM, 11:49 PM, next day 9:09 PM). (D) Overview of <i>G</i>. <i>wildpretii</i> colony, comprised of freshly settled cells (arrowhead) and large older cells with multilayered and size-increased loricae (arrow). (E) Freshly settled amoeba with multiple parietal vacuoles (arrowhead) that force the plastids into a star-like shape in the cell center. (F) Sessile cells with multiple vacuoles (black arrowhead) at the MCB periphery and smaller vesicles (white arrowhead) indicating the position of additional lorica material excretion. (G) TEM of sessile cell with multiple vesicles (arrowhead) at the cell periphery close to the existing lorica. Chloroplast with thylakoids in stacks of three (angle brackets), which can fork (white arrowhead) or pair with other thylakoid stacks. (H) TEM of sessile cell with multilayered lorica, the ostiole of which was closed during the addition of the innermost layer. (I) TEM of sessile cell with a multilayered lorica. A cross section of the reticulopodium is visible in front of the still opened ostiole. C—chloroplast, N—nucleus, O—ostiole, R—reticulopodium, L—lorica, V—vesicle. Scale bars A, C, E,F: 10 μm, B, D: 50 μm, G: 0.5 μm, H, I: 5 μm.</p

<i>Guanchochroma wildpretii</i> gen. et spec. nov. (Ochrophyta) Provides New Insights into the Diversification and Evolution of the Algal Class Synchromophyceae

<div><p>A new relative of the chrysophyte genus <i>Chrysopodocystis</i> was found in Tenerife and termed <i>Guanchochroma wildpretii</i>. This unicellular alga was most noticeably discernible from <i>Chrysopodocystis socialis</i> (the only species of this genus) by the presence of a cyst-like stage with a multilayered lorica, which also functions as a dispersal unit and shows secondary wall growth. Secondary expansion of loricae (cell casings not involved in cell division, usually with a more or less pronounced opening) has never been observed previously and marks a unique feature of the new taxon. Plastids are non-randomly distributed within cells of <i>G</i>. <i>wildpretii</i>. 18S rRNA gene analyses identified the two species as sister lineages and placed them in a monophyletic group with the Synchromophyceae, a heterokont algal (Ochrophyta) class characterized by the presence of chloroplast complexes. Yet, neither <i>Chrysopodocystis</i> nor <i>Guanchochroma</i> showed this feature in ultrastructure analyses. Additionally, their 18S rRNA genes possessed distinct inserts, the highest GC-content known for Ochrophyta and exceptionally long branches on the Ochrophyta 18S rDNA phylogenetic tree, suggesting substantially increased substitution rates along their branch compared to Synchromophyceae. Plastid marker data (<i>rbc</i>L) recovered a monophyletic clade of <i>Chrysopodocystis</i>, <i>Guanchochroma</i> and Synchromophyceae as well, yet with lower supports for internal split order due to limited resolution of the marker. Evidence for the sequence of events leading to the formation of the plastid complex of Synchromophyceae still remains ambiguous because of the apparently short timeframe in which they occurred.</p></div

Arrangement of plastids in <i>Guanchochroma wildpretii</i>.

<p>(A) TEM of two amoebae in one multilayered lorica after binary division. The nucleolus is distinct within the nucleus. Vacuoles, mostly roundish, are present in different sizes. The plastids show no pyrenoids and are not interconnected. (B) TEM of an amoeba within a multilayered lorica. Vacuoles are present, yet with highly variable outline. Some plastids show terminal elongations without thylakoids (arrowhead). No Nucleus is visible in this section plane. (C) TEM of an amoeba within a multilayered lorica, showing four plastids and a nucleus without nucleolus in the section plane. Plastids possess tubular to globular shaped protrusions (white arrowhead) that group in the cell center (black arrowhead). The cytoplasm appears highly granular with variably shaped, non-globular vesicles. V—vesicle, N—nucleus, L—lorica, C chloroplast. Scale bars 5 μm.</p

Morphology of <i>Chrysopodocystis socialis</i>.

<p>(A) TEM of sessile amoeba encased in a thin lorica. Central nucleus surrounded by multiple vacuoles. Parietal chloroplasts are pressed against the cell membrane. (B) TEM of one amoeba in a lorica during cytokinesis with a diaphragm-like structure (arrowhead). (C) TEM of migrating amoeba moving between two loricae. Chloroplasts are flattened against the cell membrane. (D+E) Migrating amoeba with leading and trailing filopodium. Chloroplasts are randomly distributed in the cell and the nucleus positioned in the center. Sessile amoebae encased in globular, hyaline loricae (arrows). After binary division, one daughter cell (asterisk) leaves the lorica through the ostiole (arrowhead). In some focus planes, the short rim of the ostiole is visible. (F) TEM of two sessile amoebae in one lorica after binary division. (G) Sequence of lorica formation from establishment of multiple vacuoles (left, 9:55 PM), the fusion into one vacuole (center, 10:30 PM), to the complete reduction of vacuoles and the re-establishment of a connection to the meroplasmodium (right, next day 4:45 PM). The final lorica is considerably larger than the MCB, leaving room (asterisk) for expansion during cell growth. Scale bars A, F: 5 μm, B, C: 2 μm, D, E, G: 10 μm. C—chloroplast, N—nucleus, L—lorica, V—vesicle.</p

mTOR complex protein expressions and rictor phosphorylation at Ser1235.

<p>Cardiomyocytes were pretreated for 30 min with 20 nM rapamycin and then stimulated with 10 nM IGF-1 in presence or absence of 10 nM E2 for 24 h. A, shown are representative western blots for mTOR, rictor and raptor and B,C,D, quantitative analysis with mean ± SEM of fold stimulation by IGF-1 of at least 3 independently performed experiments (B,C,D). * p < 0.05, **p < 0.0095. Exposure to rapamycin lead to downregulation of mTOR and rictor, which were more pronounced under culture conditions without E2. E, Western blots for GSK3β-pS9, GSK3β, rictor pS1235 and rictor and F,G,H,I quantitative analysis of at least 3 independently performed experiments indicate that rictor phosphorylation at S1235 by GSK-3β which has been reported to interfere with Akt-substrate binding to mTORC2, thereby downregulating mTORC2 activity. IGF-1 induced strong phosphorylation of GSK-3β at Ser9 in the absence and presence of E2, however, rapamycin pretreatment only reduced this increased phosphorylation in E2 co-treated cardiomyocytes, indicating increased activity of GSK-3β. This higher activity was not associated with increased phosphorylation of rictor at S1235.</p

Rapamycin does not impair E2 induced ERK activation.

<p>A, ERK phosphorylation was assessed by immunoblots from lysates of HL-1 cells cultured in the presence of 10 nM E2 and treated with 20 nM rapamycin and IGF-1 for 24 h. B, Results from densitometric analysis of blots and determination of IGF-1 induced increase in protein phosphorylation levels from at least 3 independently performed experiments. C, Immunoblots and D, E, densitometric analyses of cardiomyocytes with MEK1/2 inhibition by 1μM PD 184352 1 h prior to IGF-1 stimulation resulted in increased mTORC2 activity as indicated by increased Akt-pS473 in E2 cotreated cells. * p < 0.04,** p < 0.007, *** p < 0.0007. F, Inhibition of Erk phosphorylation by MEK1/2 inhibitor PD 184352 did not inverse rapamycin effect on Akt-pS473 in E2 cultured cardiomyocytes as investigated by western blotting and G-I, Densitometric analyses; mean ± SEM of fold stimulation by IGF-1 is shown of at least 3 independently performed experiments. * p < 0.05.</p

Sexual dimorphism of mTORC2 activation in response to rapamycin <i>in vivo</i>.

<p>Male and female C57Bl/6J mice were treated with rapamycin (“low concentration” 1.5 mg/kg, administered i.p. every third day) or vehicle (control) for 42 days (n = 6-8/group). mTORC2 activity was assessed by assessment of Akt phosphorylation at S473 and its nuclear localization. A, representative western blots are shown for Akt-pS473, Akt and GAPDH of mice treated as described above. B shows results from quantitative analysis of western blots for Akt-pS473 normalized to Akt (mean ± SEM; * p < 0.035) from female and male mouse hearts. C, Immunostaining for Akt-pS473 of male and female cardiac tissue sections treated with either vehicle (control) or rapamycin for 42 days and D, quantification of % cardiomyocyte nuclei stained for Akt-pS743 (mean ± SEM; *** p < 0.0001). Male mice had lower basal mTORC2 activity, yet responded to rapamycin with an increase in phosphorylation of Akt at S473, associated with increased nuclear localization important for induction of cardioprotective mechanisms. In contrast, female mice responded to rapamycin with reduced phosphorylation of Akt at S473 and loss of cardioprotective nuclear Akt.</p

SERCA2A expression is regulated by mTORC2.

<p>Rictor silencing was induced by cell transfection with rictor siRNA and then, cells were stimulated with IGF-1 for 24 hours in presence or absence of E2. mTORC2 downregulation was confirmed by abolished Akt-pS473 and resulted in decreased SERCA2A protein expression. Akt-pT308, mTOR and raptor were not negatively affected by rictor silencing. A, Representative blots and B-G, quantitative analysis of three independently performed experiments are shown as mean ± SEM. * p < 0.043.</p