m brevicollis (ATCC)
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ATCC
m brevicollis
M Brevicollis, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/m brevicollis/product/ATCC
Average 90 stars, based on 17 article reviews
M Brevicollis, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/m brevicollis/product/ATCC
Average 90 stars, based on 17 article reviews
m brevicollis - by Bioz Stars,
2026-01
90/100 stars
Images
1) Product Images from "STING mediates immune responses in the closest living relatives of animals"
Article Title: STING mediates immune responses in the closest living relatives of animals
Journal: eLife
doi: 10.7554/eLife.70436
Figure Legend 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).
Techniques Used:
Figure Legend 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.
Techniques Used: Immunofluorescence, Diagnostic Assay, Staining, Software, Bacteria, Fluorescence, Expressing, Activity Assay
Figure Legend Snippet: P. aeruginosa deletion strains.
Techniques Used: Staining
Figure Legend Snippet: M. brevicollis response to P. aeruginosa factors.
Techniques Used: Membrane, Extraction
Figure Legend 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.
Techniques Used: Control, Labeling, RNA Sequencing, Western Blot, Comparison, Software
Figure Legend 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.
Techniques Used: Quantitative RT-PCR, Control, Software, Cell Culture, Bacteria, Sequencing, Generated
Figure Legend 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.
Techniques Used: Binding Assay, Sequencing, Generated, Produced, Western Blot, Control, Comparison, Software
Figure Legend 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.
Techniques Used: RNA Sequencing, Quantitative Proteomics, Control, Quantitative RT-PCR
Figure Legend 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.
Techniques Used: Western Blot, Transfection, Expressing, Software, Quantitative Proteomics, Control, RNA Sequencing, Inhibition
Figure Legend 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.
Techniques Used: Software, RNA Sequencing, Quantitative Proteomics, Control, Sequencing, Generated
Figure Legend Snippet:
Techniques Used: Knock-Out, Transfection, Construct, Isolation, Transgenic Assay, Generated, Sequencing
