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First published online 29 August 2007
doi: 10.1242/dev.000802

Development 134, 3495-3505 (2007)
Published by The Company of Biologists 2007
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Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans

Cornelia Schmutz, Julia Stevens and Anne Spang*

Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.


Figure 1
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  		Fig. 1. The RhoGAP protein K09H11.3 is required to control membrane ruffling in the early C. elegans embryo. (A) Pictures from time-lapse studies of wild-type and K09H11.3 RNAi-treated embryos. K09H11.3 RNAi treatment was performed by feeding dsRNA-expressing bacteria targeting the RhoGAP domain containing the N-terminus of the K09H11.3 gene (rga-3). Embryos are grown at 20°C and mounted for microscopy: anterior ruffling is more pronounced in the RNAi-treated embryo (lower panel) than in the wild-type embryo (upper panel). This exaggerated ruffling also persists longer in the RNAi-treated embryo than in wild type. (B) Quantification of ruffles in embryos up to pronuclear meeting (PNM) in wild type (n=8) and after K09H11.3 RNAi treatment (n=13). The ruffles were counted from time-lapse movies of individual embryos. The average number of ruffles per embryo at a given time point is indicated. (C) Schematic drawing of different RhoGAP-domain-containing proteins in humans (hs), Xenopus laevis (Xl), C. elegans (Ce) and Saccharomyces cerevisiae (Sc). The RhoGAP domain (green) is present in most RhoGAPs in the more C-terminal part of the protein. RGA-3 and RGA-4 belong to a family of uncharacterized RhoGAP-domain proteins, which carry the GAP domain at the N-terminus of the protein. The SH2 domain, the C1 domain and the CRAL/Trio domain are depicted in red, blue and yellow, respectively. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Knockdown of either RGA-3 or RGA-4 leads to a hyper-contractile cortex. (A) Schematic drawing of the RNAi constructs used: for rga-3, apart from the initially cloned N-terminal sequence, constructs matching the middle part and to the 3'UTR of the gene are chosen; for rga-4, an RNAi targeting the middle part of the gene is also created. Exons are indicated in white stretches; introns in yellow. Introns are bigger in the Y75B7AL.4 gene (rga-4) compared to K09H11.3 (rga-3). (B) Pictures of Nomarski time-lapse studies after feeding with bacteria expressing dsRNA of the different RNAi constructs. For all constructs, a membrane-ruffling phenotype was observed. s, seconds. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3. RGA-3 and RGA-4 are RhoGAPs for RHO-1 and not for CDC-42. (A-D) Epistasis RNAi experiments of rho-1 (A), cdc-42 (B), ect-2 (C) or let-502 (D) with or without rga-3 and rga-4 (rga-3/4) were performed by feeding with the RNAi construct in question either alone, or by mixing together the same amounts of bacteria containing the rga-3/4 construct or RNAi construct being studied and feeding these at the same time. In the case of LET-502, epistasis experiments were also performed with the let-502(ok1283) allele, which is a knockout of LET-502. (A) Concomitant knockdown of RGA-3/4 and RHO-1 led to a catastrophic one-cell arrest, similar to rho-1 RNAi treatment [rho-1(RNAi)]. (B) cdc-42(RNAi) did not rescue the rga-3/4(RNAi) phenotype. The knockdown of the RhoGEF ECT-2 (C) and the Rho-associated kinase LET-502 (D) rescued the rga-3/4(RNAi) phenotype. Nuclei were visualized with GFP::H2B. Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. GFP::RHO-1 accumulates at ingressing furrows in rga-3/4(RNAi) embryos. (A) Pictures from time lapses showing GFP::RHO-1 in wild-type or rga-3/4(RNAi) embryos. The different stages are depicted in the cartoon. (B) Visualization of GFP intensities in the respective embryos using ImageJ. Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. Hyper-contractility in rga-3/4(RNAi) embryos is dependent on NMY-2, and NMY-2::GFP is enriched at cortical membrane ruffles in rga-3/4(RNAi) embryos. (A) nmy-2(RNAi) rescues the membrane-ruffling defect of rga-3/4(RNAi). All three proteins were knocked down at the same time by feeding. (B) Time-lapse images of NMY-2::GFP behaviour in wild-type and rga-3/4(RNAi) embryos. NMY-2::GFP is strongly enriched in the pseudo-cleavage furrow and at the anterior cortex after rga-3/4(RNAi). (C) Quantification of the fluorescence intensity of NMY-2::GFP using ImageJ. (D) Quantification of the length of the NMY-2::GFP domains at the plasma membrane in wild-type (n=7) and rga-3/4(RNAi) (n=9) zygotes. Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. Polarity establishment is not affected in rga-3/4(RNAi) embryos but the anterior PAR-6-domain size fluctuates more in rga-3/4(RNAi) embryos compared with wild type. (A) GFP::PAR-2 and (B) GFP::PAR-6 localization is determined in vivo in wild-type and in rga-3/4(RNAi) embryos. GFP::PAR-2 as well as GFP::PAR-6 is correctly localized in rga-3/4(RNAi) embryos at the posterior and anterior cortex, respectively. (C) The relative size of the GFP::PAR-2 (wild type: n=24 embryos; RNAi: n=18) and GFP::PAR-6 (wild type: n=22; RNAi: n=25) domains was determined by drawing a line from the anterior to the posterior end of the embryo and then measuring the size of the individual domain. The size of the GFP::PAR-6 domain fluctuated more in rga-3/4(RNAi) embryos, compared with wild type. The variations in the domain sizes are statistically significant (P<0.01). The size of the GFP::PAR-2 domain was less affected. Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. CYK-4 and RGA-3/4 fulfil different functions in the early C. elegans embryo. To compare the functions of the three RhoGAPs - RGA-3/4 and CYK-4 - present in the early embryo, RNAi epistasis experiments were performed by injecting dsRNA into the embryos. Whereas CYK-4 was essential for the first division and to recruit GFP::PAR-2 to the posterior cortex (upper panels), RGA-3/4 played a role in downregulating contraction (middle panels). Simultaneous knockdown of all three RhoGAPs resulted in a combination of both phenotypes; embryos did not undergo cytokinesis, the cortex remained contractile and NMY-2 spread over the entire cortex (lower panels). Scale bar: 20 µm.

 

Figure 8
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  		Fig. 8. rga-3/4(RNAi) causes strong gonad defects in let-502(sb106) mutants. (A) Gonad arms were extracted from worms and stained with rhodamin-phalloidin and DAPI. The gonads are oriented with the distal tip pointing towards the left. A schematic drawing of the gonad is included. The arrowheads point to the rachis formation defects. The arrow points to the `hole' in the let-502(sb106) rga-3/4(RNAi) gonad. (B) A gonad from the let-502(ok1283) mutant, which represents a knock-out allele, was stained as in A. The arrow points to the `hole'. (C) Quantification of gonad defects of N2 and let-502(sb106) animals treated with rga-3/4 or cyk-4 dsRNA. The number of gonads used for quantification of a particular phenotype is given in parenthesis. Depending on how the gonads adhered to the glass surface, not all phenotypes could be quantified in the same gonad. Ambiguous-looking gonads were not included in the analysis. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2007