Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization

Figures

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  Figure 1.. GRAF depletion leads to hyper constriction of contractile rings during cellularization. (A) GRAF genomic locus is shown with UTR, introns and exons. Grafi, GrafgRNA target the exon 1 region, and Graf2i targets the exon 10 region. GRAF protein (1025aa) contains a BAR, PH, RhoGAP and SH3 domain. GrafCR57 has a stop codon at amino acid 28 and is predicted to form a 27aa peptide. (B) Schematic depiction of cellularization in the Drosophila embryo with plasma membrane (black), nuclei (gray) and contractile ring (red) organization at the base of the furrow. The base of the furrow where the contractile ring assembles is hexagonal at the early stage, circular at the mid stage and constricted at the late stage. GrafCR57 mutant embryos show premature ring formation in the early stage and hyper constriction in mid and late stages with nuclei getting squeezed to show a bottleneck phenotype. (C–E) GRAF depletion leads to hyper constricted contractile rings in cellularization. (C) Phalloidin (gray)-labeled furrow tip sections show polygonal organization (white arrowhead) in control embryos, nanos-Gal4; Graf i shows circular organization, and GrafCR57 shows ring constriction (white arrowhead)…

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  Figure 1—figure supplement 1.. Phylogenetic tree of GRAF across various species. (A) The amino acid sequences for Drosophila melanogaster GRAF and its orthologs from Schizosaccharomyces cerevisiae (RGA2), Saccharomyces pombe (rga2), Dictyostelium discoideum (gacJJ), Caenorhabditis elegans (T04C9.1), Danio rerio (Arhgap42a), Xenopus laevis (Arhgap26), Xenopus tropicalis (Arhgap26), mouse (Arhgap42) and humans (Arhgap26) amino acid sequence were obtained from UniPort software. These 12 protein sequences were aligned in the NCBI multiple protein blast using the neighbor-joining method. The Drosophila GRAF protein is highlighted in yellow. (B) The domains present in each species are marked in each of the 12 proteins. Multicellular organisms from C. elegans to humans in the list contain a conserved BAR, PH, RhoGAP and SH3 domain from the N terminus to the C terminus. Bar represents the number of substitution events per site.

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  Figure 1—figure supplement 2.. GRAF knockdown phenotypes and the Crispr-Cas9 strategy for generating a GRAF null mutant. (A) Graf2i and Grafgrna expressing embryos enhanced ring constriction. Phalloidin (gray)-stained control embryos in early cellularization show polygonal organization of the furrow tip, whereas Graf2i and GrafgRNA, nanos-Cas9 show rings. In mid cellularization, control embryos show circular rings and Graf2i and GrafgRNA; nanos-Cas9 expressing embryos show constricted rings. In late cellularization, control embryos (n = 54 embryos) show constricted rings, and Graf2i (95.23% showed enhanced constriction in cellularization, n = 21 embryos) and GrafgRNA; nanos-Cas9 (89.18% showed enhanced constriction in cellularization, n = 37 embryos) embryos hyper constricted rings. (B) Graf null mutant generation using the Crispr-Cas9 strategy. Females containing the nanos-Cas9 transgene were crossed to males containing a transgene that ubiquitously expresses GrafgRNA against exon 1. The F1 generation males containing the nanos-Cas9 and GrafgRNA were crossed with homozygous FM7a virgin females. Exon 1 of the Graf gene will get cleaved by Cas9 during spermatogenesis and be subjected to DNA repair by the…

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  Figure 2.. GRAF protein is enriched at the furrow in mid cellularization. (A–C) GRAF localizes at the furrow tips in cellularization. (A) Control embryos (n = 36 embryos) immunostained with GRAF (red) and Dlg (green) show GRAF enrichment at the furrow tip in early and mid cellularization (white arrowhead shows edge enrichment, whereas yellow arrowhead shows curved region with weaker signal). GRAF is decreased from the furrow tip in late cellularization. GrafCR57 (100%, n = 11) embryos show depletion of GRAF antibody in cellularization. (B) Quantification of GRAF antibody fluorescence intensity as cortex to cytosol ratio shows increased staining in mid cellularization as compared to early and late cellularization (n = 25 rings, 5 per embryo, 5 embryos per stage, 25 embryos) (furrow length used for early = 4–6 µm, mid = 7–11 µm, late = 17–31 µm). (C) GrafCR57 (furrow length range: 3–24 µm) shows loss of GRAF antibody staining intensity compared to controls (furrow length range: 5–25 µm) (n = 30 rings, 5 per embryo, 6 embryos). Data is represented as mean ± s.d. ***p<0.001, two-tailed Mann–Whitney test. (D, E) GRAF-GFP (D, green) colocalizes with Sqh-mCherry (D, red) in early and mid…

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  Figure 2—figure supplement 1.. GRAF knockdown shows reduction in GRAF antibody staining in cellularization. (A) Grafi shows decrease in GRAF antibody staining compared to control embryos (64%, n = 14 embryos). (B) Quantification of GRAF antibody fluorescence intensity as cortex to cytosol ratio shows reduction of intensity in Grafi (furrow length range: 3–20 µm) compared to controls (furrow length range: 3–14 µm) (n = 55 rings, 5 per embryo, 11 embryos each). Data is represented as mean ± s.d. ***p<0.001 (two-tailed Mann–Whitney test). Scale bars: 5 µm. Figure 2—figure supplement 1—source data 1.Plotted values for fluorescence intensity analysis.

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  Figure 3.. Graf mutant embryos show hyper contractility during early and mid cellularization. Laser ablation was performed using line at the furrow tip during early and mid cellularization. (A, E) Representative images show control and GrafCR57 mutant embryos with the region before, at 0 s (marked with yellow dotted line) and 70 s after ablations (recoil region marked with yellow dotted line) in early and mid cellularization, respectively. (B, F) Quantifications of ring displacement after laser ablation during early and mid phase (control:black line, n = 5 embryos; GrafCR57 mutant:red line, n = 5 embryos each). (C,D,G,H) Scatter plot showing maximum displacement (C, G) of ring and initial recoil velocity (D, H) after laser ablation during early and mid phase (n = 5 embryos each). Data is represented as mean ± s.d.**p<0.01, ***p<0.001, two-tailed Mann–Whitney test. Figure 3—source data 1.Plotted values for displacement and initial recoil velocity.

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  Figure 4.. RhoGAP domain of GRAF is essential for ring constriction in cellularization. (A–D) AnillinRBD-GFP (A, C, green) colocalizes with Sqh-mCherry (red) in early, mid and late cellularization in controls. In GrafCR57; AnillinRBD-GFP, the signal is more spread as compared to in between adjacent rings. Representative images from one of n = 3 embryos of controls and GrafCR57 are shown. (B, D) A yellow bar depicted in the Sqh-mCherry image is used for the estimation of the intensity line profile. (E) Schematic showing full-length GRAF protein with GFP and GRAFΔRhoGAP-GFP proteins. (F) GrafCR57 and GrafCR57;GRAFΔRhoGAP-GFP embryos expressing Sqh-mCherry show hyper constriction compared to controls and Graf CR57; GRAF-GFP (yellow arrowhead highlights furrow tip at all stages). (G) Quantification shows a significantly lower ring area for Graf CR57, Graf CR57;GRAFΔRhoGAP-GFP as compared to controls and Graf CR57; GRAF-GFP expressing Sqh-mCherry (n = 15 rings, 5 rings per embryo, 3 embryos). Data is represented as mean ± s.d. ***p<0.001, one-way ANOVA, repeated measure with Dunnett’s multiple comparison test, the Graf CR57 and GrafCR57;GRAFΔRhoGAP-GFP mutant curves are statistically different…

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  Figure 4—figure supplement 1.. GrafCR57;GRAFΔRhoGAP-GFP recruitment dynamics. (A) Snapshots from live imaging of GrafCR57;GRAF-GFP and GrafCR57;GRAFΔRhoGAP-GFP show that GrafCR57;GRAFΔRhoGAP-GFP in aggregates in early cellularization followed by a furrow tip recruitment (seen in the form of rings) in mid cellularization and loss at the furrow in late cellularization (yellow arrowhead denotes furrow tip). (B) Quantification of fluorescence intensity relative to the maximum shows higher intensity for GrafCR57;GRAFΔRhoGAP-GFP at early stages as compared to GrafCR57;GRAFΔRhoGAP-GFP (n = 15 rings, 5 rings per embryo, 3 embryos). Data is represented as mean ± s.d. *p<0.05, two-tailed Mann–Whitney test. Figure 4—figure supplement 1—source data 1.Plotted values for fluorescence intensity analysis.

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  Figure 5.. GRAF overexpression shows inhibition of ring constriction in cellularization. (A–C) Schematic (A) shows Graf gene and the Graf overexpression line, GrafEP09461 containing a P-element (with a UAS site) insertion in 5′UTR in an orientation to drive the downstream Graf gene. (B) GRAF-OE crossed to mat-Gal4 shows an increase in GRAF (100%, n = 19) when stained with GRAF (red) and Dlg (green) (control n = 24 embryos). (C) Quantification shows an increased GRAF antibody fluorescence in GRAF-OE (furrow length range: 7–19 µm) embryos compared to controls (furrow length range: 7–20 µm) (n = 25 rings, 5 per embryo, 5 embryos each). (D) Sqh-mCherry images and (E) area quantification show a significantly higher ring area in GRAF-OE as compared to controls (yellow arrowhead marks the furrow tip) (n = 15 rings, 5 per embryo, 3 embryos)(control values were repeated from Figure 3 for comparison). Data is represented as mean ± s.d. **p<0.01, ***p<0.001, two-tailed Mann–Whitney test. Scale bars: 5 µm. Figure 5—source data 1.Plotted values for fluorescence intensity and ring area analysis.

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  Figure 6.. Graf-depleted embryos show increased Myosin II in cellularization, whereas GRAF overexpression shows reduced Myosin II. (A–D) Fixed images (A) of control and GrafCR57 embryos (100%, n = 31 embryos) stained with Dlg (green) and Zipper (red) showing higher Zipper intensity in early (white arrowhead shows Zipper foci), mid (white arrowhead shows Zipper enrichment in control and spreading in GrafCR57 mutant) and late cellularization (control n = 16 embryos). (B) Cortex to cytosol ratio of Zipper antibody fluorescence shows a significant increase in GrafCR57 as compared to controls (n = 30 rings, 10 per embryo, 3 embryos each). (C) Control, GRAF-OE and GrafCR57 embryos expressing Sqh-mCherry (gray) are shown in early (yellow arrowhead marks Sqh-mCherry foci), mid (yellow arrowhead marks Sqh-mCherry enrichment) and late cellularization (yellow arrowhead in sagittal sections marks the furrow tip). (D) Quantification of Sqh-mCherry in late cellularization (yellow region marked in Figure 6—figure supplement 1B) in GRAF-OE shows reduction in intensity, whereas Graf CR57 and Graf CR57;GRAFΔRhoGAP-GFP shows higher intensity as compared to controls. Graf CR57; GRAF-GFP shows a rescue in…

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  Figure 6—figure supplement 1.. Sqh-mCherry recruitment and furrow length analysis in GrafCR57 and GRAF-OE. (A) Furrow length quantified from live imaging with Sqh-mCherry for controls, GrafCR57 and GRAF-OE shows a similar trend of the slow and fast phase of ingression in GRAF-OE as compared to controls. The slow phase is extended in GrafCR57, and the final length is achieved at a later time in the fast phase (n = 15 furrow lengths, 5 furrows per embryo, 3 embryos). Data is represented as mean ± s.d. ***p<0.001; one-way ANOVA, repeated measure with Dunnett’s multiple comparison test, the GrafCR57 is statistically different from control and GRAF-OE. (B) Quantification of Sqh-mCherry fluorescence in GRAF-OE embryos (each time point between 3 and 18 min is significantly different from controls and GrafCR57) shows a delay in Sqh-mCherry enrichment at the furrow as compared to controls. GrafCR57 embryos (each time point between 30 to 41 min is significantly different from controls) show sustained Sqh-mCherry in late cellularization as compared to controls (n = 15 rings, 5 per embryo, 3 embryos). Data is represented as mean ± s.d. ***p<0.001. Two-way ANOVA, Bonferroni post-tests where each genotype compared to…

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  Figure 7.. GRAF overexpression-driven loss of ring constriction is suppressed by RhoGEF2 overexpression in cellularization. (A–B) (A) The increased constriction seen in RhoGEF2-OE embryos is suppressed in GRAF-OE similar to controls when imaged live with Sqh-mCherry (gray) (yellow arrowhead marks furrow tip in sagittal sections, white arrowhead marks Sqh-mCherry foci in RhoGEF2-OE) and (B) when stained with phalloidin (B, GRAF-OE 100% shows loss of constriction, n = 50 embryos, RhoGEF2-OE 96.15% shows enhanced constriction, n = 52 embryos, GRAF-OE; RhoGEF2-OE 57.69% shows constriction comparable to controls, n = 52 embryos). (C) Quantification of contractile ring area from Sqh-mCherry shows decreased area in RhoGEF2-OE, increased area in GRAF-OE and normal area in GRAF-OE; RhoGEF2-OE as compared to controls (n = 15 rings, 5 rings per embryo, 3 embryos) (control values were repeated from Figure 3 and GRAF-OE values were repeated from Figure 4 for comparison). Data is represented as mean ± s.d. ***p<0.001, one-way ANOVA, repeated measure with Dunnett’s multiple comparison test, the GRAF-OE, RhoGEF2-OE is statistically different from controls and GRAF-OE; RhoGEF2-OE. (D) Quantification of…