Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 Å resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 Å resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix

Conservation of structure and function in vertebrate c-FLIP proteins despite rapid evolutionary change

Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development

Les Amours des dieux, ballet héroïque, [parole de Fuzelier], mis en musique par M. Mouret, représenté pour la 1re fois par l'Académie Royale de Musique le mardi 16 septembre 1727

Titre uniforme : Mouret, Jean-Joseph (1682-1738). Compositeur. [Les amours des dieux]Comprend : P 1-41 - Prologue - P. 1-47 - Neptune et Amymone (1ère entrée) - P. 1-87 - Jupiter et Niobé (2e entrée) - P. 1-75 - Apollon et Coronis (3e entrée) - P. 1-59 - Ariane et Bacchus (4e entrée) - Fol 1 et 2 - Air ajouté aux amours de Dian

AIG1 affects in vitro and in vivo virulence in clinical isolates of <i>Entamoeba histolytica</i> - Fig 6

<p><b>EHI_176590-HA expression in KU27</b> (A) Immuno-detection of EHI_176590-HA ectopically expressed in strain KU27. Total cell lysates, from mock-transfected and EHI_176590-HA-expressing strain KU27 trophozoites, were analyzed by immunoblot analysis with anti-EHI_176590 (left panel) and anti-HA antibody (right panel). The arrow indicates EHI_176590-HA, and asterisks indicate protein bands detected by non-specific immunoreactivity. (B) Low-magnification confocal immunofluorescence images of EHI_176590-HA-expressing and mock-transfected KU27. Trophozoites were fixed and reacted with anti-Igl and anti-HA antibodies. Merged fluorescent (IGL/HA) and DIC images are also shown. (C) Adhesion of the parental, EHI_176590-HA-expressing, and mock-transfected KU27 to HRBCs. Parental (open bars), EHI_176590-HA-expressing (black bars), and mock-transfected (gray bars) KU27 strains were co-cultured with HRBCs on ice for 30 min, and adherent HRBCs per ameba were counted. The total number of trophozoites was set to 100%, and the percentage of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of four biological replicates.</p

Localization of EHI_175690, and protrusion formation on EHI_175690-HA-expressing amoebae.

<p>(A) Immunofluorescence image, obtained by confocal microscopy, showing localization of EHI_176590. EHI_176590-HA-expressing amoebae were fixed and reacted with anti-HA antibody. (B) Magnified image of the inset in (A). Arrows indicate protrusions. (C) Immunofluorescence image, obtained by confocal microscopy, showing localization of EHI_176590-HA and the intermediate subunit of galactose/N-acetylgalactosamine-specific lectin (Igl). EHI_176590-HA-expressing amoebae were fixed and stained with anti-HA and anti-Igl antibodies. Arrow indicates protrusion. Islet showing signal-enhanced image to highlight the protrusion. (D) Effect of EHI_176590-HA expression on the percentage of amoebae that possess protrusions. EHI_176590-HA-expressing and mock transformants (HA) were immunostained as in (C) and the number of cells with protrusions, longer than 0.7 μm, was counted and are presented as percentages. The averages of three independent experiments, in which 100–200 amoebae were counted, are shown. (E) Effect of EHI_176590-HA expression on the number of protrusions per amoeba. EHI_176590-HA-expressing and mock transformants (HA) were immunostained as in (C), and the number of protrusions, per amoeba, was counted. The averages of three independent experiments, in which 100–200 amoebae were counted, are shown. * indicates p<0.01.</p

Verification of the loss of EHI_176590 in KU27, overexpression of EHI_176590, and phenotypes caused by EHI_176590 overexpression.

<p>(A) Verification by PCR of the lost genomic region, containing the EHI_176590 gene, in KU27. (B) Immuno-detection of EHI_176590 in KU27 and KU50. Total cell lysates, from the indicated strains, were analyzed by immunoblot analysis with anti-EHI_176590 antibody. The arrow depicts the EHI_176590 protein, which is present in KU50, but missing in KU27; asterisk indicates the protein band detected by non-specific binding or cross reaction. (C) Immuno-detection of EHI_176590-HA. Total lysates from EHI_176590-HA-expressing and mock transformants were analyzed by immunoblot analysis with an anti-HA antibody (left panel) or anti-EHI_176590 antibody (right panel). Black arrowheads indicate full-length EHI_176590-HA, and gray arrowheads indicate minor truncated forms of EHI_17690-HA. The arrow indicates intrinsic EHI_176590. Asterisks depict protein bands detected by non-specific binding or cross reaction. (D) Low magnification confocal immunofluorescence images of the EHI_176590-HA-expressing and mock-transfected transformants. Trophozoites were fixed and reacted with anti-Igl and anti-HA antibodies. Merged fluorescent (IGL/HA) and differential interference contrast (DIC) images are also shown. (E) Increased adherence to HRBCs by EHI_176590 overexpression. EHI_176590-HA-expressing (filled bars) and mock (open bars) transformants were co-cultured with HRBCs on ice for 30 min, and the number of adherent HRBCs per ameba were counted. The total number of trophozoites was set to 100% and the percentages of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of four biological replicates (2292 and 1762 trophozoites were counted for the EHI_176590-HA-expressing and mock transformants, respectively). **p-value <0.01, ***p-value <0.001 (F) Cell motility of the EHI_176590-HA-expressing and mock-transfected transformants. Time-lapse images of CellTracker Green-loaded EHI_176590-HA-expressing and mock (“HA”) transformants were analyzed by ICY software to measure cell motility. The average speed and standard deviation of the motility of EHI_176590-HA and mock vector transformants (HA) are shown; 135 and 106 trophozoites, respectively, were monitored in four independent experiments.</p

Liver abscess formation in hamsters challenged with EHI_176590 gene-silenced and EHI_176590-HA-expressing transformants using animal-passaged virulent strain Cl6.

<p>(A) Confirmation of EHI_176590 gene silencing by immunoblot analysis. Total lysates, from EHI_176590 gene-silenced and mock transformants, were subjected to immunoblot analysis using anti-EHI_176590 (upper panel) and control anti-CS1 antibodies (lower panel). (B) Effect of EHI_176590 gene silencing on liver abscess formation. Approximately 1×10⁶ trophozoites of EHI_176590 gene-silenced and mock-transfected strains (created from animal-passaged virulent Cl6) were injected into the livers of hamsters. Injected animals were sacrificed 6 days post-infection, and the liver and abscesses were dissected and weighed separately. Averaged percentages of the weight of abscesses per liver are shown. *p<0.004. (C) Adhesion of EHI_176590 gene-silenced and mock-transfected strains to HRBCs. Mock-transfected (black bars) and EHI_176590 gene-silenced (white bars) strains were co-cultured with HRBCs on ice for 30 min, and adherent HRBCs per ameba were counted. The percentage of trophozoites, bound to 0–5, 6–10, or >10 HRBCs, are shown. Error bars indicate standard deviations of three biological replicates. (D) Confirmation of EHI_176590-HA expression by immunoblot analysis. Total lysates from EHI_176590-HA and mock transformants were analyzed by immunoblot with anti-HA, anti-EHI_176590, and anti-CS1 antibodies (upper, middle, and lower panels, respectively). (E) Effect of EHI_176590-HA overexpression on liver abscess formation. Approximately 3×10⁶ trophozoites, of EHI_176590-HA and mock-transfected transformants, were injected into the liver of hamsters, and liver abscess formation was evaluated as in (B). **p<0.003. (F) Adhesion of EHI_176590-HA and mock-transfected strains to HRBCs. Trophozoites of mock-transfected (black bars) and EHI_176590-HA transfected (white bars) strains were co-cultured with HRBCs, and adhesion was evaluated as in (C).</p

Comparative genome analysis of <i>E</i>. <i>histolytica</i> strains KU27 and KU50.

<p><b>(</b>A) RPKM score of the genomes of strains KU27 and KU50. The X-axis represents 8,163 annotated genes of <i>E</i>. <i>histolytica</i> HM-1:IMSS in the order of locus tag gene ID. (B) Gene copy number variations in KU27 and KU50. RPKM scores of 3252 genes, with a higher value than the RPKM median of all genes, are shown. The 181 genes that are present at higher copy number in KU50, compared to that in KU27, are displayed as red dots; the 37 genes, present in higher copy number in KU27 compared to that in KU50, are shown as blue dots. The x-axis represents 8,163 annotated <i>E</i>. <i>histolytica</i> HM-1:IMSS genes ordered by locus tag gene ID. The y-axis represents the number of gene copies, per gene, expressed as KU50 gene copy number minus KU27 gene copy number. (C, D) Raw mapping data of KU27 and KU50 reads to the regions that contain genes present in KU50, but absent in KU27 (C), and those present in KU27, but absent in KU50 (D). <i>E</i>. <i>histolytica</i> HM-1:IMSS reference genome sequence was used for mapping. The y-axis displays mapping read coverage.</p