The RhoGAP domain of CYK-4 has an essential role in RhoA activation

Abstract

Cytokinesis in animal cells is mediated by a cortical actomyosin-based contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both nonmuscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2]. ECT-2, the guanine nucleotide exchange factor (GEF) that activates RhoA during cytokinesis, is regulated by phosphorylation and subcellular localization [3-5]. ECT2 localization depends on interactions with CYK-4/MgcRacGAP, a Rho GTPase-activating protein (GAP) domain containing protein [5, 6]. Here we show that, contrary to expectations, the Rho GTPase-activating protein (GAP) domain of CYK-4 promotes activation of RhoA during cytokinesis. Furthermore, we show that the primary phenotype caused by mutations in the GAP domain of CYK-4 is not caused by ectopic activation of CED-10/Rac1 and ARX-2/Arp2. However, inhibition of CED-10/Rac1 and ARX-2/Arp2 facilitates ingression of weak cleavage furrows. These results demonstrate that a GAP domain can contribute to activation of a small GTPase. Furthermore, cleavage furrow ingression is sensitive to the balance of contractile forces and cortical tension.

Figure 1. RHO-1/RhoA is a dosage-sensitive regulator of cytokinesis

C. elegans embryos depleted of RHO-1. The extent of RHO-1 depletion was classified phenotypically (mild: pseudocleavage failure; moderate: pseudocleavage and second division failure; and severe: pseudocleavage, first and second cell divisions failure). (A) Kymographs of the equatorial region of representative GFP::PH expressing embryos. (B) Kinetics of furrow ingression (n=7 embryos for mild, n=7 for severe, n=20 for moderate, n=30 for control). (C) Cortical myosin accumulation in the furrow region (n = 8 for mild, n = 5 for severe, n = 6 for moderate, n = 11 for control). Error bars represent s.e.m. and scale bar is 10 µm.

Figure 2. CED-10/Rac1 depletion allows cyk-4(or749) embryos to complete furrow ingression without restoring furrow ingression rate nor cortical myosin

(A) Kymographs of the equatorial region of representative GFP::PH expressing embryos of the indicated genotypes. (B) Quantitation of the kinetics of furrow ingression of embryos of the indicated genotypes. Error bars represent s.e.m. (n> 24 embryos for all genotypes). (C) Cortical myosin (NMY-2::GFP) at a central plane of embryos of the indicated genotypes at various time points following anaphase onset. (D) Quantitation of the amount of cortical myosin of embryos of the indicated genotypes (n≥ 9 embryos for all genotypes). (E) Box and whisker plot of the amount of equatorial cortical f-actin (GFP::MOE) of embryos of the indicated genotypes (n≥10 embryos for each genotype; p<.02, Mann-Whitney U-test). (F) Box and whisker plot of the amount of equatorial cortical anillin (GFP::ANI-1) of embryos of the indicated genotypes (n=10 embryos for each genotype; p<.02, Mann-Whitney U-test). Error bars represent s.e.m. and scale bar is 10 µm.

Figure 3. Mutations in the GAP domain of CYK-4 abrogate central spindle dependent furrowing, independent of the activation of RAC-1/Rac1

(A) Control [>20, 100%], _Gα(RNAi) [11, 100%], zyg-9(RNAi) [14, 100%], cyk-4(or749) [>20, 100%], cyk-4(or749);Gα(RNAi) [8, 12.5%]; cyk-4(or749);zyg-9(RNAi) [18, 0%] embryos expressing GFP::PH and mCherry::H2B are shown at the indicated times after anaphase onset. Gα(RNAi) refers to co-depletion of GOA-1 and GPA-16. Numbers in brackets represent number of embryos and the percent of embryos with ingressing cleavage furrows. (B) Nomarski images of representative embryos of the indicated genotypes are shown at the indicated times after anaphase onset. (n ≥ 9 for all genotypes, 100% embryos exhibited the phenotype shown). Scale bar 10 µm.

Figure 4. Mutation of ced-10/rac1 and depletion of ARX-2/Arp2 induce cortical instability and improve cytokinesis in ect-2(-) embryos

(A) Kymographs of the anterior region of the central plane of embryos expressing GFP::PH for three control, arx-2(RNAi), and ced-10(n1993) embryos from anaphase onset. (B) The extent of cortical instability was quantitated by subtracting sequential frames (40 planes, every 10 s after anaphase onset) and measuring the area of the subtracted image (see methods for details). (C) Kymograph analysis of the behavior of MOE::GFP labeling f-actin beginning 90 s after anaphase onset. Note the overall reduction of cortical actin and the transient behavior of remaining f-actin (arrowheads) in arx-2(RNAi) embryos. (D) The progression of cytokinesis in embryos of the indicated genotypes at the restrictive temperature (n=34, 15, and 11 embryos, respectively). Colors represent four bins representing the observed extents of furrow ingression. Scale bars 10 µm.