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. 2. OSGN-1 regulates RhoA activity at the C. elegans PGC intercellular bridge. (A) Time-lapse images (sum projections of three confocal slices) of the C. elegans P4 germline founder cell division in embryos depleted of control (empty L4440 vector), osgn-1, rho-1 or rga-3/4 by RNAi. Embryos express markers for RhoA activity (GFP::AHPH, green), membrane (mCh::PLC∂-PH, magenta), and chromatin (mCh::H2B, magenta). Time (in min) is relative to anaphase onset (A.O.). (Scale bar, 3 μm.) (B) Fluorescence levels over time of the GFP::AHPH reporter measured at the equatorial region (cytokinetic furrow/ intercellular bridge) of P4 in embryos depleted of control (black), osgn-1 (blue), rho-1 (orange), or rga-3/4 (green) by RNAi, as in panel A. Values correspond to mean ± SEM over three biological replicates (n = 5 to 19 embryos in total per condition, as indicated). (C) Average fluorescence intensity of the GFP::AHPH reporter measured during P4 cytokinetic furrow ingression (6 to 9 min after A.O.) and PGC intercellular bridge maintenance (36 to 45 min after A.O.) in embryos depleted of the indicated gene products. Values represent individual embryos. Bars denote average ± SD (n = 5 to 19 embryos, as depicted). ns = non-significant, *P < 0.05, ***P < 0.001, two-way ANOVA followed by a Šídák multiple comparison test.

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, Control, Plasmid Preparation, Membrane, Fluorescence, Comparison

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

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. 4. C. elegans OSGN-1 localizes to the intercellular bridge in dividing HeLa cells. (A) Western blot analysis (revealed with anti-GFP antibodies) of extracts from HeLa cells untransfected (mock) or expressing either GFP-HA, GFP-HA-OSGN-1, or GFP-HA-OSGIN1 (here and below denoted with C. elegans (Ce) and H. sapiens (Hs) prefixes for clarity). (B) Representative indirect immunofluorescence images of HeLa cells stably expressing GFP-HA-OSGN-1 (Top set) or GFP-HA-OSGIN1 (Bottom set) and stained with antibodies against GFP (green) and RhoA (magenta) at the indicated mitotic stages. Hoechst labels nuclei (blue). Arrows denote the intercellular bridge. (Scale bar, 10 μm.) (C and D) Time-lapse images (C) and ratio of bridge/cytoplasm GFP fluorescence signal at telophase (D) of HeLa cells stably expressing H2B-mRFP (blue) and GFP-HA (Top row) GFP-HA-OSGN-1 (Middle row) or GFP-HA-OSGIN1 (Bottom row). Arrows denote the intercellular bridge and Insets depict 1.6× magnification of the bridge in the last frame. Time (in min) is relative to anaphase onset. (Scale bar, 10 μm.) The values in (D) represent individual cells and bars denote average ± SD, ns = nonsignificant *P < 0.05, ***P < 0.001, Kruskal–Wallis followed by a Dunn’s multiple comparison test.

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: Western Blot, Expressing, Immunofluorescence, Stable Transfection, Staining, Fluorescence, Comparison