m brevicollis  (ATCC)

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: P. aeruginosa influences M. brevicollis motility. Movies depicting M. brevicollis cultures after exposure to E. coli or P. aeruginosa bacteria for 16 hours. In the absence of pathogenic bacteria,M. brevicollis is a highly motile flagellate and swims up in the water column (Movie 1). However, co-culturing M. brevicollis with P. aeruginosa results in reduced motility and cell settling (Movie 2).

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques:

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Immunofluorescence illuminates the diagnostic cellular architecture of M. brevicollis , including an apical flagellum ( f ) made of microtubules, surrounded by an actin-filled microvilli feeding collar (co). Staining for tubulin (green) also highlights cortical microtubules that run along the periphery of the cell body, and staining for F-actin (magenta) highlights basal filopodia (fp). DNA staining (blue) highlights the nucleus ( n ). ( B ) M. brevicollis exhibits truncated flagella after exposure to P. aeruginosa. M. brevicollis were exposed to E. coli or P. aeruginosa for 24 hr, and then fixed and immunostained. Arrows point to flagella. Green: anti-tubulin antibody (flagella and cell body), magenta: phalloidin (collar), blue: Hoechst (bacterial and choanoflagellate nuclei). Scale bars represent 10 μm. Flagellar length was quantified using Fiji, and statistical analysis (unpaired t-tests) was performed in GraphPad software. ( C ) Exposure to P. aeruginosa , but not other Gammaproteobacteria, results in M. brevicollis cell death. Bacteria were added to M. brevicollis culture at an MOI of 1.5 (at Hours = 0), and M. brevicollis cell density was quantified at indicated time points. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software; p - values shown are from comparisons between Flavobacterium and P. aeruginosa . ( D–F ) M. brevicollis does not ingest P. aeruginosa bacteria. ( D,E ) M. brevicollis were fed either fluorescent E. coli ( D ) or P. aeruginosa ( E ) for 1 hr, and then visualized by DIC (D,E, left) and green fluorescence (D, E, right). Fluorescent food vacuoles were observed in choanoflagellates fed E. coli, but not P. aeruginosa . ( F ) M. brevicollis was exposed to GFP-expressing E. coli , V. parahaemolyticus , C. jejuni , or P. aeruginosa (MOI = 50) for 1 hr, and then imaged by DIC and green fluorescence to quantify number of cells with internalized bacteria. Choanoflagellate cells with ≥1 GFP+ food vacuole were scored as GFP+, and cells without any GFP+ food vacuoles were scored as GFP–. Data represent cells quantified over three biological replicates. ( G,H ) P. aeruginosa does not broadly inhibit M. brevicollis phagocytosis. ( G ) Internalization of 0.2 μm fluorescent beads was used to quantify phagocytic activity after exposure to E. coli or P. aeruginosa bacteria. Although cells did not phagocytose P. aeruginosa, cells exposed to E. coli and P. aeruginosa had similar phagocytic uptake of beads. Data represent n = 600 cells from three biological replicates. Statistical analyses (multiple unpaired t-tests) were performed in GraphPad software. ( H ) Exposure to P. aeruginosa does not inhibit phagocytic uptake of E. coli . Internalization of fluorescent E. coli or P. aeruginosa bacteria was quantified after exposure to unlabeled P. aeruginosa (PAO1 strain). Data represent n = 200 cells from two biological replicates. Statistical analysis (unpaired t-test) was performed in GraphPad software. ( I ) Secreted P. aeruginosa molecules are sufficient to induce M. brevicollis cell death. 5 % (vol/vol) bacterial conditioned medium was added to M. brevicollis culture (at Hours = 0), and M. brevicollis cell density was quantified at indicated time points. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software, and p- values shown are from comparisons between Flavobacterium and P. aeruginosa . ( J ) Sustained exposure to secreted P. aeruginosa molecules is required to induce M. brevicollis cell death. P. aeruginosa or Flavobacterium conditioned medium (5% vol/vol) was added to stationary-phase M. brevicollis cultures. After indicated times, cultures were washed and resuspended in fresh media. M. brevicollis cell density was quantified after 24 hr. The % survival is a measure of the cell density of P. aeruginosa -treated cells relative to Flavobacterium -treated controls. Data represent mean ± SD for three biological replicates.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Immunofluorescence, Diagnostic Assay, Staining, Software, Bacteria, Fluorescence, Expressing, Activity Assay

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: P. aeruginosa deletion strains.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Staining

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: M. brevicollis response to P. aeruginosa factors.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Membrane, Extraction

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Volcano plot displaying genes differentially expressed between M. brevicollis exposed to P. aeruginosa PAO1 and Flavobacterium (control) conditioned medium for three hours. Differentially expressed genes are depicted by blue (674 upregulated genes) and yellow (232 downregulated genes) dots (fold change ≥2; FDR ≤ 1e –4 ). Select genes that are upregulated or may function in innate immunity are labeled. RNA-seq libraries were prepared from four biological replicates. ( B ) After a 3-hr treatment, STING mRNA levels (determined by RNA-seq) increase 1.42-fold in cells exposed to Flavobacterium conditioned medium and 5.54 fold in cells exposed to P. aeruginosa conditioned medium, compared to untreated controls. ( C ) STING protein levels increase after exposure to P. aeruginosa . STING levels were examined by immunoblotting at indicated timepoints after exposure to Flavobacterium or P. aeruginosa conditioned medium (5% vol/vol). Tubulin is shown as loading control, and intensity of STING protein bands were quantified relative to tubulin. Statistical analysis (one-way ANOVA, Dunnett’s multiple comparison) was performed in GraphPad software, and p - values shown are calculated using 0 hr timepoint as the control group.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Control, Labeling, RNA Sequencing, Western Blot, Comparison, Software

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Gene ontology enrichmentanalysis of genes identified as differentially expressed (fold change≥2; FDR≤1e -4 ) afterexposure to P. aeruginosa . Due to lack of annotation, >40% of the differentiallyexpressed genes were not included in the enrichment analysis. ( B ) qRT-PCR validationof STING mRNA after exposure to Flavobacterium or P. aeruginosa conditioned mediafor 3 hr, compared to vehicle control. Error bars represent SD. Statisticalanalysis (t-test) was performed in GraphPad software ( C ) To validate the M. brevicollis STING antibody, cell lysates from M. brevicollis were immunoblotted alongside celllysates from S. rosetta , a closely-related choanoflagellate species that does not have aSTING homolog. A band at 36kD, the predicted size of M. brevicollis STING, is detectable in M. brevicollis lysate but not S. rosetta lysate. Arrow indicates STING band.Non-specific bands are likely due to co-cultured feeding bacteria. Tubulin is shown asloading control. ( D ) Protein sequence alignment (generated by Clustal Omega multiplesequence alignment) of M. brevicollis and animal STING proteins, colored by similarity. M. brevicollis STING and human STING are 19.1% identical and 36.6% similar at the amino acid level.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Quantitative RT-PCR, Control, Software, Cell Culture, Bacteria, Sequencing, Generated

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Schematic of choanoflagellate ( M. brevicollis ), sea anemone ( N. vectensis ), insect ( D. melanogaster ), and mammalian ( M. musculus and H. sapiens ) STING proteins. Transmembrane (TM) domains are depicted in gray, STING cyclic dinucleotide binding domain (CDN) in purple, and C-terminal tail domain (CTT) in blue. ( B ) Partial protein sequence alignment (generated by Clustal Omega multiple sequence alignment) of M. brevicollis and animal STING proteins, colored by similarity. M. brevicollis STING and human STING are 19.1 % identical and 36.6 % similar at the amino acid level. Key cyclic dinucleotide-interacting residues from human STING structure are indicated by circles. ( C ) Dose-response curves of M. brevicollis exposed to cyclic dinucleotides for 24 hr reveal that treatment with 2’3’cGAMP, but not 3’3’ cGAMP, c-di-AMP, or c-di-GMP, leads to M. brevicollis cell death in a dose-dependent manner. Data represent mean ± SD for at least three biological replicates. ( D ) STING protein levels increase after exposure to 2’3’cGAMP, but not bacterially produced cyclic dinucleotides. M. brevicollis STING levels were examined by immunoblotting 5 hr after exposure to 2’3’cGAMP (100 µM), 3’3’cGAMP (200 µM), c-di-GMP (200 µM), or c-di-AMP (200 µM). Tubulin is shown as loading control, and intensity of STING protein bands were quantified relative to tubulin. Shown is a representative blot from three biological replicates. Statistical analysis (one-way ANOVA, Dunnett’s multiple comparison) was performed in GraphPad software. ( E ) STING protein levels increase and remain elevated after exposure to 100 µM 2’3’cGAMP. Tubulin is shown as loading control, and data are representative of three biological replicates. Statistical analysis (one-way ANOVA, Dunnett’s multiple comparison) was performed in GraphPad software, and p - values shown are calculated using 0 hr timepoint as control group.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Binding Assay, Sequencing, Generated, Produced, Western Blot, Control, Comparison, Software

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A,B ) Volcano plots displaying RNA-seq differential expression analysis of M. brevicollis treated with ( A ) 100 µM 2’3’cGAMP or ( B ) 200 µM 3’3’cGAMP for 3 hr, relative to an untreated control. Genes with a fold change ≥2 and false discovery rate ≤10e –4 are depicted by black dots. STING is highlighted in red. RNA-seq libraries were prepared from three (2’3’ cGAMP) or two (3’3’ cGAMP) biological replicates. ( C ) M. brevicollis STING mRNA levels increase in response to 2’3’cGAMP. (Left) RNA-seq fold change of STING mRNA after exposure to 100 µM 2’3’cGAMP or 200 µM 3’3’cGAMP for 3 hr, compared to vehicle control. (Right) qRT-PCR fold change of STING mRNA after exposure to 100 µM 2’3’cGAMP or 200 µM 3’3’cGAMP for 3 hr, compared to vehicle control. ( D ) Venn diagram comparing the overlap of genes identified as differentially expressed after treatment with 2’3’cGAMP, 3’3’cGAMP, and P. aeruginosa (DEG cutoff: fold change ≥3, false discovery rate ≤10e –4 ). ( E,F ) Representative immunostained M. brevicollis demonstrating 2’3’cGAMP stimulates the formation of STING puncta at perinuclear regions. M. brevicollis was left untreated ( E ), or exposed to 100 µM 2’3’cGAMP ( F ) for 5 hr. Cells were fixed and STING levels and localization were probed using an anti-STING antibody. ( E’,F’ ) Exposure to 2’3’cGAMP results in increased numbers of STING puncta compared to untreated controls. ( E’’,F’’ ) Z-slice images of the plane containing the nucleus ‘n’ show that STING puncta localize to perinuclear regions. Green: anti-tubulin antibody (flagella and cell body), magenta: anti-STING antibody, blue: Hoechst. Scale bar represents 2 µm.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: RNA Sequencing, Quantitative Proteomics, Control, Quantitative RT-PCR

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) The genotypes of wild type and genome-edited STING – strains at the STING locus. ( B ) STING protein is not detectable by immunoblot in STING – cells. Shown is a representative blot from three biological replicates. ( C,D ) STING is necessary for 2’3’cGAMP-induced cell death. ( C ) Wild type and STING – strains were treated with increasing concentrations of 2’3’cGAMP, and survival was quantified after 24 hr. In contrast to wild type cells, 2’3’cGAMP does not induce cell death in STING – cells. Data represent mean ± SD for four biological replicates. ( D ) Wild type and STING – cells were transfected with STING-mTFP, and treated with puromycin to generate stable clonal strains. Stable expression of STING-mTFP in STING – cells partially rescued the phenotype of 2’3’cGAMP-induced cell death. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software. ( E ) Wild type and STING – strains have distinct transcriptional responses to 2’3’ cGAMP. Differential expression analysis was performed on wild type and STING – cells treated with 100 µM 2’3’cGAMP or a vehicle control for 3 hr. A heatmap comparing the log 2 fold change of genes identified as differentially expressed (FC ≥2; FDR ≤ 10 –4 ) in wild-type cells after 2’3’ cGAMP treatment, to their log 2 fold change in STING – cells after 2’3’ cGAMP treatment. RNA-seq libraries were prepared from two biological replicates. ( F ) Presence of STING in the transcriptomes of diverse choanoflagellate species. Data from . ( G ) Effects of 2’3’cGAMP on different choanoflagellate species. Choanoflagellates were grown to late-log phase, and treated with increasing concentrations of 2’3’cGAMP. Survival was quantified after 24 hr. 2’3’cGAMP only affected the survival of M. brevicollis and S. macrocollata , the two sequenced choanoflagellate species with a STING homolog. Data represent mean ± SD for three biological replicates. ( H ) Wild type and STING – cells have similar survival responses to LPS, suggesting that STING is not required for mediating a response to LPS. Wild type and STING – strains were treated with increasing concentrations of E. coli LPS, and survival was quantified after 24 hr. Data represent mean ± SD for four biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software. ( I,J ) STING renders M. brevicollis more susceptible to P. aeruginosa -induced growth inhibition. ( I ) Wild type and STING – cells were exposed to control Flavobacterium or P. aeruginosa conditioned medium (5% vol/vol), and cell densities were quantified at indicated time points. Data represent mean ± SD for three biological replicates. ( J ) Percent survival calculated from growth curves in ( I ). Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Western Blot, Transfection, Expressing, Software, Quantitative Proteomics, Control, RNA Sequencing, Inhibition

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Sanger sequences of the consensus genotype at the site of gene editing in wild type and STING – cells. STING – cells have a seven base-pair deletion that leads to premature stop codons. ( B ) Growth curves of wild type and STING – cells indicate that both strains have similar growth dynamics. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software. ( C ) Volcano plot displaying RNA-seq differential expression analysis of STING – cells treated with 100 µM 2’3’cGAMP for 3 hr, relative to an untreated control. Genes with a fold change ≥2 and false discovery rate ≤10e –4 are depicted by black dots. RNA-seq libraries were prepared from two biological replicates. ( D ) Venn diagram comparing the overlap of genes identified as differentially expressed (FC ≥3; FDR ≤ 10 –4 ) after treatment with 2’3’cGAMP in wild type and STING – cells. ( E ) Protein sequence alignment (generated by Clustal Omega multiple sequence alignment) of STING proteins from choanoflagellates S. macrocollata and M. brevicollis and animals, colored by similarity.

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Software, RNA Sequencing, Quantitative Proteomics, Control, Sequencing, Generated

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet:

Article Snippet: Strain, strain background ( M. brevicollis ) , M. brevicollis , ATCC PRA-258 , PMID: 18276888 , .

Techniques: Knock-Out, Transfection, Construct, Isolation, Transgenic Assay, Generated, Sequencing

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Choanoflagellates and the ancestry of neurosecretory vesicles

doi: 10.1098/rstb.2019.0759

Figure Lengend Snippet: Neurosecretory vesicle proteins in animals and their closest living relatives. (Top) Schematic model of the core molecular components of animal neurosecretory vesicles. (Below) Core proteins of animal neurosecretory vesicles can be assigned to ten categories: V-ATPases, vesicular neurotransmitter transporters, transporter and transporter-like proteins, proteins with four transmembrane domains, synapsins, synaptotagmins, secretory SNAREs, endosomal SNAREs, transiently associated proteins SNARE binding partners and co-chaperones (modified after ). Black dots indicate the presence of clear protein sequence homologues (also see electronic supplementary material, table S1), while lines indicate that a homologue was not detected in the respective organism. Taxonomic groupings are indicated as follows: brown box, Opisthokonta; red box, Holozoa; blue box, Metazoa; violet box, Bilateria; yellow box, Vertebrata. Phylogenetic tree based on a consensus phylogeny [ – ]. A. que , Amphimedon queenslandica ; B. den , Batrachochytrium dendrobatidis ; C. owc , Capsaspora owczarzaki ; D. rer , Danio rerio ; D. mel , Drosophila melanogaster ; H. sap , Homo sapiens ; M. bre , Monosiga brevicollis ; M. lei , Mnemiopsis leidyi ; N. vec , Nematostella vectensis ; R. ory , Rhizopus oryzae ; S. cer , Saccharomyces cerevisae ; S. pur , Strongylocentrotus purpuratus ; S. ros , Salpingoeca rosetta ; T. adh , Trichoplax adhaerens . B. den , R. ory and S. cer are fungi. * = protein of interest-like, a = putative SLC17A5-homologue, b = domain structure lost.

Article Snippet: Digital image stacks of the TEM sections of M. brevicollis and S. rosetta were imported into AMIRA (FEI Visualization Sciences Group) and aligned semi-manually.

Techniques: Binding Assay, Modification, Sequencing

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Choanoflagellates and the ancestry of neurosecretory vesicles

doi: 10.1098/rstb.2019.0759

Figure Lengend Snippet: Synaptobrevin in the choanoflagellate Salpingoeca rosetta . ( a ) Domain architecture of Salpingoeca rosetta synaptobrevin and Homo sapiens synaptobrevin 1 and 2. ( b ) Sequence alignment of the SNARE motif of S. rosetta synaptobrevin and H. sapiens synaptobrevin 1 and 2. The 15 layers (highlighted in blue including layers −1 to −7 and layers +1 to +8) important for SNARE complex formation are shown. The conserved arginine residues forming the ionic 0 layer are shown in green. ( c – c ″) Apical view of an S. rosetta cell stained with antibodies against ( c ) tubulin (grey) and ( c ′) synaptobrevin (yellow). ( c″ ) Merged. ( d – d ″) Lateral view of a different S. rosetta cell stained with antibodies against ( d ) tubulin and ( d ′) synaptobrevin. ( d ″) Merged. The dashed square in ( d ″) indicates to position of ( h ). ( e – e ″) A rosette colony of S. rosetta stained with the same antibodies as in ( c ). The orange arrows indicate a basal synaptobrevin signal. ( e ) Tubulin. ( e′ ) Synaptobrevin. ( e ″) Merged. The dotted square in ( e ″) indicates the position of ( f ). ( f ) Synaptobrevin-positive vesicles are in close contact with tubulin-positive cytoskeletal filaments. ( g ) TEM image showing the close contact between apical vesicles and tubulin filaments; av, apical vesicles; tf, tubulin filaments. ( h ) Image of a 3D reconstruction of the apical region of an S. rosetta cell. Apical vesicles are coloured in orange, tubulin filaments in light grey and the soma in half-transparent grey. Close contacts of vesicles and cytoskeletal filaments are indicated with white asterisks. The scale bar is 1 µm. Sros, Salpingoeca rosetta ; Hsap, Homo sapiens .

Article Snippet: Digital image stacks of the TEM sections of M. brevicollis and S. rosetta were imported into AMIRA (FEI Visualization Sciences Group) and aligned semi-manually.

Techniques: Sequencing, Staining

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Choanoflagellates and the ancestry of neurosecretory vesicles

doi: 10.1098/rstb.2019.0759

Figure Lengend Snippet: The diverse vesicular landscape of choanoflagellates. ( a ) Images of a 3D reconstruction of all vesicles in M. brevicollis (left) and S. rosetta (right). Individual vesicles are coloured randomly, and the cell is shown in half-transparent grey. A plot of all vesicle diameters measured is given in the middle. Mean diameters of different vesicle types are indicated by triangles in the same colours as in ( b–l ). ( b–f ) Visualization of separated vesicles of each vesicle type to show the localization within the soma of M. brevicollis . The Golgi-apparatus is shown in half-transparent lilac in ( b ). TEM images showing each vesicle type are given beneath images of the 3D model ( b′ – f′ ). ( g – k ) Visualization of separated vesicles of each vesicle type to show the localization within the soma of S. rosetta . The Golgi-apparatus is shown in half-transparent lilac in ( b ). TEM images showing each vesicle type are given beneath the 3D models ( g′ – k′ ). Scale bars of TEM images are 50 nm, scale bars of images of 3D reconstructions are approximately 250 nm. ( l ) Box and whiskers plots of the vesicle diameters within the different vesicle types (also see electronic supplementary material, table S2 and video S1 and S2). M. brevicollis : Golgi-associated vesicles (minimum: 37; median: 54; maximum: 79); small vesicles (minimum: 43; median 72; maximum 104); apical vesicles (minimum: 81; median: 109; maximum: 161); large extremely electron-lucent vesicles (minimum: 85; median: 132; maximum: 223); large electron-dense vesicles (minimum: 108; median 124; maximum 189). S. rosetta : Golgi-associated vesicles (minimum: 32; median: 55; maximum: 87); small vesicles (minimum: 51; median 78; maximum 116); apical vesicles (minimum: 102; median: 175; maximum: 233); large extremely electron-lucent vesicles (minimum: 153; median: 202; maximum: 301); medium vesicles (minimum: 107; median 125; maximum 180). M.bre, Monosiga brevicollis ; S.ros, Salpingoeca rosetta . nu = nucleus, ga = Golgi apparatus, ER = endoplasmic reticulum, mt = mitochondria, pm = plasma membrane.

Article Snippet: Digital image stacks of the TEM sections of M. brevicollis and S. rosetta were imported into AMIRA (FEI Visualization Sciences Group) and aligned semi-manually.

Techniques: Clinical Proteomics, Membrane

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Choanoflagellates and the ancestry of neurosecretory vesicles

doi: 10.1098/rstb.2019.0759

Figure Lengend Snippet: Neurosecretory vesicle proteins in animals and their closest living relatives. (Top) Schematic model of the core molecular components of animal neurosecretory vesicles. (Below) Core proteins of animal neurosecretory vesicles can be assigned to ten categories: V-ATPases, vesicular neurotransmitter transporters, transporter and transporter-like proteins, proteins with four transmembrane domains, synapsins, synaptotagmins, secretory SNAREs, endosomal SNAREs, transiently associated proteins SNARE binding partners and co-chaperones (modified after ). Black dots indicate the presence of clear protein sequence homologues (also see electronic supplementary material, table S1), while lines indicate that a homologue was not detected in the respective organism. Taxonomic groupings are indicated as follows: brown box, Opisthokonta; red box, Holozoa; blue box, Metazoa; violet box, Bilateria; yellow box, Vertebrata. Phylogenetic tree based on a consensus phylogeny [ – ]. A. que , Amphimedon queenslandica ; B. den , Batrachochytrium dendrobatidis ; C. owc , Capsaspora owczarzaki ; D. rer , Danio rerio ; D. mel , Drosophila melanogaster ; H. sap , Homo sapiens ; M. bre , Monosiga brevicollis ; M. lei , Mnemiopsis leidyi ; N. vec , Nematostella vectensis ; R. ory , Rhizopus oryzae ; S. cer , Saccharomyces cerevisae ; S. pur , Strongylocentrotus purpuratus ; S. ros , Salpingoeca rosetta ; T. adh , Trichoplax adhaerens . B. den , R. ory and S. cer are fungi. * = protein of interest-like, a = putative SLC17A5-homologue, b = domain structure lost.

Article Snippet: M. brevicollis cultures (50154; American Type Culture Collection) were cultured in artificial seawater mixed with Wards cereal grass medium in a 1 : 1 ratio, adjusted to a salt concentration of 53 mS cm −1 and sterile filtered as previously described [ ].

Techniques: Binding Assay, Modification, Sequencing

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Choanoflagellates and the ancestry of neurosecretory vesicles

doi: 10.1098/rstb.2019.0759

Figure Lengend Snippet: The diverse vesicular landscape of choanoflagellates. ( a ) Images of a 3D reconstruction of all vesicles in M. brevicollis (left) and S. rosetta (right). Individual vesicles are coloured randomly, and the cell is shown in half-transparent grey. A plot of all vesicle diameters measured is given in the middle. Mean diameters of different vesicle types are indicated by triangles in the same colours as in ( b–l ). ( b–f ) Visualization of separated vesicles of each vesicle type to show the localization within the soma of M. brevicollis . The Golgi-apparatus is shown in half-transparent lilac in ( b ). TEM images showing each vesicle type are given beneath images of the 3D model ( b′ – f′ ). ( g – k ) Visualization of separated vesicles of each vesicle type to show the localization within the soma of S. rosetta . The Golgi-apparatus is shown in half-transparent lilac in ( b ). TEM images showing each vesicle type are given beneath the 3D models ( g′ – k′ ). Scale bars of TEM images are 50 nm, scale bars of images of 3D reconstructions are approximately 250 nm. ( l ) Box and whiskers plots of the vesicle diameters within the different vesicle types (also see electronic supplementary material, table S2 and video S1 and S2). M. brevicollis : Golgi-associated vesicles (minimum: 37; median: 54; maximum: 79); small vesicles (minimum: 43; median 72; maximum 104); apical vesicles (minimum: 81; median: 109; maximum: 161); large extremely electron-lucent vesicles (minimum: 85; median: 132; maximum: 223); large electron-dense vesicles (minimum: 108; median 124; maximum 189). S. rosetta : Golgi-associated vesicles (minimum: 32; median: 55; maximum: 87); small vesicles (minimum: 51; median 78; maximum 116); apical vesicles (minimum: 102; median: 175; maximum: 233); large extremely electron-lucent vesicles (minimum: 153; median: 202; maximum: 301); medium vesicles (minimum: 107; median 125; maximum 180). M.bre, Monosiga brevicollis ; S.ros, Salpingoeca rosetta . nu = nucleus, ga = Golgi apparatus, ER = endoplasmic reticulum, mt = mitochondria, pm = plasma membrane.

Article Snippet: M. brevicollis cultures (50154; American Type Culture Collection) were cultured in artificial seawater mixed with Wards cereal grass medium in a 1 : 1 ratio, adjusted to a salt concentration of 53 mS cm −1 and sterile filtered as previously described [ ].

Techniques: Clinical Proteomics, Membrane

Journal: Molecular Biology and Evolution

Article Title: Patterns of Ancestral Animal Codon Usage Bias Revealed through Holozoan Protists

doi: 10.1093/molbev/msy157

Figure Lengend Snippet: Whole Genome and Codon Usage Statistics in the Transcriptomes of the Three Holozoan Protists.

Article Snippet: Complete annotated transcriptome sequences for M. brevicollis and S. rosetta were downloaded from the Origins of Multicellularity Project at the Broad Institute; the C. owczarzaki transcriptome was downloaded from the EnsemblProtists database.

Techniques:

Journal: Molecular Biology and Evolution

Article Title: Patterns of Ancestral Animal Codon Usage Bias Revealed through Holozoan Protists

doi: 10.1093/molbev/msy157

Figure Lengend Snippet: Nc plots for M. brevicollis , S. rosetta and C. owczarzaki . GC3s values are shown on the x -axis and Nc values are given on the y -axis. The curved line on each plot represents the expected position of genes evolving under a neutral mutation model .

Article Snippet: Complete annotated transcriptome sequences for M. brevicollis and S. rosetta were downloaded from the Origins of Multicellularity Project at the Broad Institute; the C. owczarzaki transcriptome was downloaded from the EnsemblProtists database.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups

doi: 10.1371/journal.pone.0032867

Figure Lengend Snippet: After Walker et al. (2011). Groups labelled in black encompass the taxa listed in ; those in grey encompass the taxa in the EMBL/Genbank dbEST, and non-redundant protein and nucleotide databases. Blue = Opisthokonta, Brown = Amoebozoa, Magenta = Excavata, Green = Archaeplastida, Grey = CCTH Supergroup Red = SAR Supergroup, with stramenopile, alveolate and rhizarian labeled. Taxa in capitals contain multicellular species. Taxa highlighted contain species with nitrile hydratase genes. * = eukaryotic-type nitrile hydratase. Number of * indicates the number of subunits present. # = nitrile hydratase subunit genes that may be the result of prokaryotic contamination. Note that taxa branching from a single point represent nodes with ambiguous branching, and the eukaryotic tree is unrooted.

Article Snippet: BLASTp and tBLASTn searches of the Broad Institute Origins of Multicellularity website using the M. brevicollis nitrile hydratase protein sequence found similar sequences in the genomes of Salpingoeca rosetta , Thecamonas trahens and Sphaeroforma arctica (see ).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups

doi: 10.1371/journal.pone.0032867

Figure Lengend Snippet: This form is known to be found in M. brevicollis, S. rosetta, S. diplocostata, S. arctica, T. trahens, A. anophagefferens, F. cylindrus, B. natans and E. huxleyi. The red area denotes the beta subunit, which is located N-terminally of the alpha subunit (green region). The CTLCSC active site is located in the alpha subunit, as shown by the shaded area. The yellow area denotes the histidine-rich stretch found between the subunit domains in opisthokonts.

Article Snippet: BLASTp and tBLASTn searches of the Broad Institute Origins of Multicellularity website using the M. brevicollis nitrile hydratase protein sequence found similar sequences in the genomes of Salpingoeca rosetta , Thecamonas trahens and Sphaeroforma arctica (see ).

Techniques: