Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: OSGN-1 is a conserved flavin-containing monooxygenase required to stabilize the intercellular bridge in late cytokinesis.
doi: 10.1073/pnas.2308570121
Figure Lengend Snippet: Fig. 3. The catalytic activity of OSGN-1 is required for stability of the C. elegans PGC intercellular bridge. (A) Schematic depiction of the different amino acid substitutions encoded by some of the osgn-1 mutants used in this study. (B and C) Representative images (B) and quantification (C) of the presence (white bars) or absence (black bars) of a membrane partition between the two PGCs in embryos expressing markers for RhoA activity (GFP::AHPH, green), membrane (mCh::PLC∂-PH, magenta) and chromatin (mCh::H2B, magenta). (Scale bar, 10 μm.) n = 14 to 57 embryos, as indicated. *P < 0.05, ***P < 0.001, Fisher’s exact test. (D–F) Representative image (D) and average fluorescence intensity (E and F) of the GFP::AHPH reporter measured during P4 cytokinetic furrow ingression (6 to 9 min after anaphase onset [A.O.]; E) and PGC intercellular bridge maintenance (36 to 45 min after A.O.; F) in embryos encoding the indicated OSGN-1 variants. Values represent individual embryos. Bars denote average ± SD (n = 13 to 23 embryos, as depicted). Individual fluorescence curves are shown in SI Appendix, Fig. S3H. ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Dunnett’s multiple comparison test. (G) Coomassie staining of purified OSGN-1 variants used in the FAD-binding assay (Top image) and Ni-NTA agarose pellets following OSGN-1 variant purifications, visually denoting the presence (yellowish coloration) or absence (white) of FAD (Bottom image). (H) Extinction coefficient spectra of BSA (negative control, gray), FAD alone (5 µM, white) or FAD bound to the recombinant OSGN-1 variants indicated (WT and mutants, colored). (I) Comparative measurement of the catalytic activity of the indicated C. elegans OSGN-1-6xHis variants (1 μM final concentration each) using the DTNB-methimazole assay, in the presence of the essential co-factor NADPH. The decrease of absorbance at 412 nm over time (in min) reports on catalytic activity. Values are corrected for reactions done in the absence of methimazole and correspond to the mean ± SEM over N = 3 assays, see Materials and Methods for details.
Article Snippet: To generate the pCMV- H2B- mRFP1 construct enabling visualization of H2BmRFP in HeLa cell nuclei, the H2B- mRFP1 sequence was amplified by PCR from pLV- RFP [Addgene #26001 (39)] and inserted using Gibson assembly in place of GFP into vector pEGFP- C1 (Clonetech, a gift from S. Carréno, Université de Montréal, Montréal, Canada).
Techniques: Activity Assay, Membrane, Expressing, Fluorescence, Comparison, Staining, Purification, Binding Assay, Variant Assay, Negative Control, Recombinant, Concentration Assay
Addgene inc is a verified supplier 
![Fig. 3. The catalytic activity of OSGN-1 is required for stability of the C. elegans PGC intercellular bridge. (A) Schematic depiction of the different amino acid substitutions encoded by some of the osgn-1 mutants used in this study. (B and C) Representative images (B) and quantification (C) of the presence (white bars) or absence (black bars) of a membrane partition between the two PGCs in embryos expressing markers for RhoA activity (GFP::AHPH, green), membrane (mCh::PLC∂-PH, magenta) and chromatin (mCh::H2B, magenta). (Scale bar, 10 μm.) n = 14 to 57 embryos, as indicated. *P < 0.05, ***P < 0.001, Fisher’s exact test. (D–F) Representative image (D) and average fluorescence intensity (E and F) of the GFP::AHPH reporter measured during P4 cytokinetic furrow ingression (6 to 9 min after anaphase onset [A.O.]; E) and PGC intercellular bridge maintenance (36 to 45 min after A.O.; F) in embryos encoding the indicated OSGN-1 variants. Values represent individual embryos. Bars denote average ± SD (n = 13 to 23 embryos, as depicted). Individual fluorescence curves are shown in SI Appendix, Fig. S3H. ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by a Dunnett’s multiple comparison test. (G) Coomassie staining of purified OSGN-1 variants used in the FAD-binding assay (Top image) and Ni-NTA agarose pellets following OSGN-1 variant purifications, visually denoting the presence (yellowish coloration) or absence (white) of FAD (Bottom image). (H) Extinction coefficient spectra of BSA (negative control, gray), FAD alone (5 µM, white) or FAD bound to the recombinant OSGN-1 variants indicated (WT and mutants, colored). (I) Comparative measurement of the catalytic activity of the indicated C. elegans OSGN-1-6xHis variants (1 μM final concentration each) using the DTNB-methimazole assay, in the presence of the essential co-factor NADPH. The decrease of absorbance at 412 nm over time (in min) reports on catalytic activity. Values are corrected for reactions done in the absence of methimazole and correspond to the mean ± SEM over N = 3 assays, see Materials and Methods for details.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_2170/pm38442170/pm38442170__page4_image1.jpg)
