1. Developmental Biology

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

  1. Swati Sharma
  2. Richa Rikhy  Is a corresponding author
  1. Biology, Indian Institute of Science Education and Research, India
https://doi.org/10.7554/eLife.63535
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Cite this article
  1. Swati Sharma
  2. Richa Rikhy
(2021)
Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization
eLife 10:e63535.
https://doi.org/10.7554/eLife.63535
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10 figures, 10 videos, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
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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 …

Figure 1—source data 1

Plotted values for ring area analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
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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), Caenorhabditi…

Figure 1—figure supplement 2
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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…

Figure 2 with 1 supplement
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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 …

Figure 2—source data 1

Plotted values for fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
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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 …

Figure 2—figure supplement 1—source data 1

Plotted values for fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig2-figsupp1-data1-v2.xlsx
Figure 3
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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 …

Figure 3—source data 1

Plotted values for displacement and initial recoil velocity.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
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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 …

Figure 4—source data 1

Plotted values for fluorescence intensity and ring area analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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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 …

Figure 4—figure supplement 1—source data 1

Plotted values for fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig4-figsupp1-data1-v2.xlsx
Figure 5
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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) …

Figure 5—source data 1

Plotted values for fluorescence intensity and ring area analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
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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), …

Figure 6—source data 1

Plotted values for fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
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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. …

Figure 6—figure supplement 1—source data 1

Plotted values for furrow length and fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 1 supplement
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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 …

Figure 7—source data 1

Plotted values for ring area and fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
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RhoGEF2 overexpression retains GRAF recruitment and membrane furrow length analysis of RhoGEF2 depletion and overexpression.

(A, B) Fixed embryos (A) stained with GRAF and Dlg antibodies show increased staining of GRAF in nanos-Gal4; RhoGEF2 (RhoGEF2-OE) embryos in mid and late cellularization (100% embryos show increased …

Figure 7—figure supplement 1—source data 1

Plotted values for furrow length analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig7-figsupp1-data1-v2.xlsx
Figure 8 with 1 supplement
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GrafCR57 hyper constriction phenotype is suppressed by RhoGEF2 depletion.

(A–B) GrafCR57 (A) shows hyper constriction, RhoGEF2i shows loss of constriction and GrafCR57; RhoGEF2i combination shows ring sizes comparable to control embryos when imaged live with Sqh-mCherry …

Figure 8—source data 1

Plotted values for ring area and fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
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RhoGEF2 depletion retains GRAF recruitment.

Fixed embryos stained with GRAF and Dlg antibodies show similar GRAF staining in nanos-Gal4; RhoGEF2 RNAi expressing embryos knockdown as compared to controls (100% embryos show GRAF staining …

Figure 9 with 1 supplement
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The hyper constriction phenotype of GrafCR57 embryos is suppressed by additional depletion of Rok.

(A) GrafCR57 shows hyper constricted contractile rings, roki shows loss of ring constriction and the GrafCR57; roki combination suppression of the hyper constriction phenotype seen in GrafCR57 when …

Figure 9—source data 1

Plotted values for ring area and fluorescence intensity analysis.

https://cdn.elifesciences.org/articles/63535/elife-63535-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
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GRAF overexpression suppresses the hyper constriction phenotype in Myosin II binding subunit (MBS)-depleted embryos.
Author response image 1
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Sqh1P expression pattern during mid cellularization in GrafCR57 mutant.

Sqh1P staining revealed patchy staining with cytoplasmic signal during mid cellularization. There is no specific localization at the furrow tip in contractile rings and no observable change in the …

Videos

Video 1
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GRAF-GFP and Sqh-mCherry distribution during ring constriction in cellularization.

The video shows living embryos expressing GRAF-GFP (green, above) and Sqh-mCherry (red, below) during cellularization. There is enrichment of GRAF-GFP and Sqh-mCherry at the furrow in mid …

Video 2
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GRAF-GFP and Sqh-mCherry distribution on the furrow in sagittal sections.

Sagittal sections were extracted to show distribution of GRAF-GFP (green, above) and Sqh-mCherry (red, below) during cellularization. Note the loss of GRAF-GFP in late cellularization, whereas …

Video 3
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GrafCR57 embryos show contractile ring recoil upon ablations during cellularization.

Live imaging of Sqh-mCherry control (left) and GrafCR57 (right) containing Sqh-mCherry (gray) subjected to laser ablations (time = 0 s) at the regions marked in a yellow line during early (above) …

Video 4
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GrafCR57 embryos show enrichment of AnillinRBD-GFP and Sqh-mCherry distribution during ring constriction in cellularization.

The video shows colocalization of AnillinRBD-GFP (green, above, left) and Sqh-mCherry (red, below, left) throughout cellularization. GrafCR57 embryos show enrichment of AnillinRBD-GFP (above, right) …

Video 5
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GrafCR57 embryos expressing Sqh-mCherry show increased ring constriction during cellularization.

Live imaging of Sqh-mCherry control (above) and GrafCR57 (below) containing Sqh-mCherry (gray) at the furrow during cellularization. GrafCR57 shows enhanced constriction as compared to controls. Sum …

Video 6
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GrafCR57; GRAFΔRhoGAP-GFP embryos expressing Sqh-mCherry show enhanced ring constriction during cellularization.

Live embryos expressing GrafCR57; GRAFΔRhoGAP-GFP (green, above) and Sqh-mCherry (red, below) are shown during cellularization. GrafCR57; GRAFΔRhoGAP-GFP shows enhanced constricted ring compared to …

Video 7
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GRAF-OE embryos expressing Sqh-mCherry show inhibition of ring constriction during cellularization.

Live imaging of Sqh-mCherry control (above) and Graf-OE (below) embryo expressing Sqh-mCherry (gray) at the furrow is shown. Graf-OE leads to inhibition of ring constriction when compared to …

Video 8
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The hyper constricted ring phenotype in RhoGEF2-OE expressing embryos is suppressed in GRAF-OE; RhoGEF2-OE.

Constriction at the furrow was imaged during cellularization in RhoGEF2-OE (above) and GRAF-OE; RhoGEF2-OE (below) containing Sqh-mCherry (gray). RhoGEF2-OE shows hyper constriction in late …

Video 9
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Inhibition of ring constriction in RhoGEF2i expressing embryos is suppressed in GrafCR57; RhoGEF2i.

Constriction at the furrow was imaged during cellularization in RhoGEF2i (above) and GrafCR57;RhoGEF2i (below) embryos expressing Sqh-mCherry (gray). RhoGEF2i knockdown embryos showed inhibition of …

Video 10
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ROKi and GrafCR57; ROKi expressing embryos show inhibition of ring constriction.

ROKi (above) and GrafCR57; ROKi (below) embryos expressing Sqh-mCherry (gray) were imaged live during cellularization. ROKi embryos show inhibition of ring constriction as compared to controls (Video…

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)Canton-SBloomington Drosophila Stock CenterBDSC:1 RRID:BDSC_1
Genetic reagent (Drosophila melanogaster)nanos-Gal4Lab stock (Mavrakis et al., 2009; Mavrakis et al., 2008)
Genetic reagent (Drosophila melanogaster)w; mat67-Gal4; mat15-Gal4Girish Ratnaparkhi, IISER, Pune, India
Genetic reagent (Drosophila melanogaster)w; P{Sqh-mCherry.M}3Bloomington Drosophila Stock CenterBDSC:59024 RRID:BDSC_59024
Genetic reagent (Drosophila melanogaster)y[1] v[1];P{TRiP.HMC03427}attP40 (GRAFshRNA1)Bloomington Drosophila Stock CenterBDSC:51853 RRID:BDSC_51853
Genetic reagent (Drosophila melanogaster)y[1] v[1]; P{TRiP.GL01207}att P40 (myosin binding subunit, MBS shRNA)Bloomington Drosophila Stock CenterBDSC:41625 RRID:BDSC_41625
Genetic reagent (Drosophila melanogaster)y[1] sc[*] v[1] sev[21]; P{TRiP.HMS01118}a ttP2 (RhoGEF2 shRNA)Bloomington Drosophila Stock CenterBDSC:34643 RRID:BDSC_34643
Genetic reagent (Drosophila melanogaster)y[1] w[*];P{UASpT7.RhoGEF2}5 (RhoGEF2 overexpression)Bloomington Drosophila Stock CenterBDSC:9386 RRID:BDSC_9386
Genetic reagent (Drosophila melanogaster)ubi-GFP::AnillinRBD/TM3Thomas Lecuit, IBDM, France, Munjal et al., 2015
Genetic reagent (Drosophila melanogaster)UASp-rok-shRNA (roki)Tony Harris, University of Toronto, Canada, Zhang et al., 2018
Genetic reagent (Drosophila melanogaster)y[1] w[67]c[23] P{EPgy2}Graf[EY094 61] (GRAF overexpression)Bloomington Drosophila Stock CenterBDSC:17571 RRID:BDSC_17571
Genetic reagent (Drosophila melanogaster)w;mat67 Spider-GFP-Sqh-mcherry/TM3serEric F. Wieschaus, Princeton University, USA, Martin et al., 2009
Genetic reagent (Drosophila melanogaster)y[1] sc[*] v[1] Graf[CR57]/FM7a (GrafCR57)This study (Richa RIkhy, IISER, Pune, India)
Genetic reagent (Drosophila melanogaster)y[1] sc[*] v[1] sev[21]; P{TKO.GS00762}att P40Bloomington Drosophila Stock CenterBDSC:76993 RRID:BDSC_76993
Genetic reagent (Drosophila melanogaster)P{KK102763}VIE-260B (GRAF shRNA2, Graf2i)Vienna Drosophila Stock Center, Vienna, Austriav110812
Genetic reagent (Drosophila melanogaster)y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=nosCas9.R}attP40Bloomington Drosophila Stock CenterBDSC:78781 RRID:BDSC_78781
Genetic reagent (Drosophila melanogaster)[w]*;p[UASp-GRAF-EGFPG1] attp40/cyoThis paper (Richa Rikhy, IISER, Pune, India)
Genetic reagent (Drosophila melanogaster)[w]*;p[UASp-GRAFΔRhoGAP-GFP] attp40/cyoThis paper (Richa Rikhy, IISER, Pune, India)
Sequence-based reagentGRAF_pUASp_Homo_Kpn1_GRAF(start)_FP (1)This paper (Sigma-Aldrich)PCR primersCCGCATAGGCCACTAGTGGATCTGGTACCATGGGCGGCGGCAAAAATGTACG
Sequence-based reagentGRAF(end)_serine linker_GFP(start)_FP (2)This paper (Sigma-Aldrich)PCR primersACTATGTGGAACATTTGAAGCCGCACCATTCCTCGAGCTCCAGCATGGTGAGCAAGGGCGAGGAGCT
Sequence-based reagentGRAF(end)_serine linker_GFP(start)_RP (3)This paper (Sigma-Aldrich)PCR primersAGCTCCTCGCCCTTGCTCACCATGCTGGAGCTCGAGGAATGGTGCGGCTTCAAATGTTCCACATAGT
Sequence-based reagentGFP_pUASp_Homo_RP (4)This paper (Sigma-Aldrich)PCR primersAACGTTCGAGGTCGACTCTAGAGGATCCTTACTTGTACAGCTCGTCCATGCCGAGAGTGAT
Sequence-based reagentRhoGAP_del_RP (5)This paper (Sigma-Aldrich)PCR primersCCTTCGTGGCGTCCGGCAACTTTGCCTCGCTGACTTTGATTTTGCCG
Sequence-based reagentRhoGAP_del_FP (6)This paper (Sigma-Aldrich)PCR primersCGGCAAAATCAAAGTCAGCGAGGCAAAGTTGCCGGACGCCACGAAGG
Sequence-based reagentGRAF FL AB vecOH FP (7)This paper (Sigma-Aldrich)PCR primersACGAAAATCTGTATTTCCAAGGCATGGGCGGCGGCAAAAATGT
Sequence-based reagentVect GRAF FL ABOH RP (8)This paper (Sigma-Aldrich)PCR primersACATTTTTGCCGCCGCCCATGCCTTGGAAATACAGATTTTCGT
Sequence-based reagentVect GRAF FL ABOH FP (9)This paper (Sigma-Aldrich)PCR primersAACATTTGAAGCCGCACCATTGGTCTCATCCTCAGTTCGA
Sequence-based reagentGRAF FL AB vecOH RP (10)This paper (Sigma-Aldrich)PCR primersTCGAACTGAGGATGAGACCAATGGTGCGGCTTCAAATGTTCCAC
Sequence-based reagentGRAF mutant scr1 FP (11)This paper (Sigma-Aldrich)PCR primersGTAAATGTTGCAAACACCGCAGTTTTCTCGAAACTCAACC
Sequence-based reagentGRAF mutant scr RP (12)This paper (Sigma-Aldrich)PCR primersTACTACTTACTCTTTGCCGCACTCATAAGATCTTTGACCT
Sequence-based reagentPot2 212 CtoT FP (13)This paper (Sigma-Aldrich)PCR primersCAATTGGCCATTTTACTTAACGATTTT
Sequence-based reagentPot2 212 CtoT RP (14)This paper (Sigma-Aldrich)PCR primersAAAATCGTTAAGTAAAATGGCCAATTG
Sequence-based reagentPot2 420 AtoG FP (15)This paper (Sigma-Aldrich)PCR primersAAGAAAACAAAAAGAAATTTGATAAAA
Sequence-based reagentPot2 420 AtoG RP (16)This paper (Sigma-Aldrich)PCR primersTTTTATCAAATTTCTTTTTGTTTTCTT
Sequence-based reagentPot2 468 delT FP (17)This paper (Sigma-Aldrich)PCR primersGCAGGAGCGTTTCCTCAATATGTC
Sequence-based reagentPot2 468 delT RP (18)This paper (Sigma-Aldrich)PCR primersGACATATTGAGGAAACGCTCCTGC
AntibodyAnti-GRAF (rabbit polyclonal)BiokloneThis study1:500; pre-absorbed usingGrafCR57 null mutant embryos
AntibodyAnti-Dlg (mouse monoclonal)DSHB4F3, RRID:AB_528203IF (1:100)
AntibodyAnti-Zipper (rabbit polyclonal)Thomas Jeffrey, Texas Tech University, TX, USA, Chougule et al., 2016IF (1:200)
AntibodyGoat anti-mouse IgG Alexa 488Molecular ProbesA-1100, RRID:AB_2534069IF (1:1000)
AntibodyGoat anti-rabbit IgG Alexa 488Molecular ProbesA-11008, RRID:AB_143165IF (1:1000)
AntibodyGoat anti-mouse IgG Alexa 568Molecular ProbesA-11004, RRID:AB_141371IF (1:1000)
AntibodyGoat anti-rabbit IgG Alexa 568Molecular ProbesA-1101, RRID:AB_143157IF (1:1000)
AntibodyHoechst 33258Molecular ProbesH-3569IF (1:1000)
AntibodySlow Fade GoldMolecular ProbesS-36937
AntibodyPhalloidin Alexa 488Molecular ProbesA-12379, RRID:AB_2315147IF (1:100)
AntibodyPhalloidin Alexa 568Molecular ProbesA-12380, RRID:AB_2759224IF (1:100)
AntibodyPhalloidin Alexa 647Molecular ProbesA-22287, RRID:AB_2620155IF (1:100)
Software, algorithmImageJ and FijiNational Institutes of Health, (Rueden et al., 2017; Schindelin et al., 2012; Schneider et al., 2012)
Software, algorithmGraphPad Prism 5Prism

Additional files

Supplementary file 1

Drosophila recombinants and crosses.

The stocks and recombinants used in this study are numbered in Table 1. The crosses carried out with the stocks for each figure for live imaging and fixed imaging of embryos along with the temperature at which each cross is carried out are detailed in Table 2.

https://cdn.elifesciences.org/articles/63535/elife-63535-supp1-v2.docx
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