In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.

Summary Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth.

ABSTRACT. Nonmuscle myosin II activity is regulated by phosphorylation of the myosin II regulatory light chain (MRLC) at Ser19 or at both Thr18 and Ser19, and the phosphorylation of MRLC promotes the contractility and stability of actomyosin. To analyze the states of MRLC phosphorylation at the leading edge in the motile HeLa cells, we have examined the subcellular distribution of monophosphorylated or diphosphorylated form of MRLC using a confocal microscope. The cross-sectional imaging revealed that monophosphorylated MRLC distributed throughout the cortical region and the leading edge, but its fluorescent signal was much stronger at the leading edge. This distribution pattern of monophosphorylated MRLC was almost identical to those of myosin II and Factin. On the other hand, diphosphorylated MRLC is localized at the base of leading edge, spatially very close to the substrate, and colocalized with F-actin in part at the base of filopodia. Diphosphorylated MRLC was hardly detectable at the tip of filopodia and the cell cortical region, where monophosphorylated MRLC was clearly detected. These localization patterns suggest that myosin II is activated at the leading edge, especially at the base but not the tip of filopodia in motile cells. Next, we analyzed the cells expressing GFP-tagged recombinant MRLCs. Expression of GFP-tagged diphosphorylatable and monophosphorylatable MRLCs led to a significant increase in the filopodial number, compared with the cells expressing nonphosphorylatable MRLC. This result indicated that expression of phosphorylatable MRLC enhances the formation of filopodia at the wound edge.Key words: myosin II regulatory light chain/phosphorylation/subcellular localization/confocal laser microscopy/wound healing/leading edge Myosin II is one of the best characterized of actin-based motor proteins. The formation and maintenance of stress fibers are believed to require myosin II activity (Chrzanowska-Wodnicka and Burridge, 1996). Nonmuscle myosin II activity is regulated by phosphorylation of the 20-kD myosin II regulatory light chain (MRLC), and the phosphorylation of MRLC promotes the contractility and stability of actomyosin (Sellers, 1991;Trybus, 1991). Monophosphorylation of MRLC at Ser19 results in a marked increase in both the stability of myosin II filaments and the activity of actin-activated myosin II Mg-ATPase. Diphosphorylation of MRLC at both Thr18 and Ser19 results in increasing the actin-activated Mg-ATPase activity, which is higher than that results from monophosphorylation of MRLC (Ikebe and Hartshorne, 1985;Itoh et al., 1992).Biochemical analyses have revealed that the level of phosphorylation of nonmuscle MRLC increases concomitantly with the contraction of cells after stimulation with serum or certain drugs (Giuliano et al., 1992;Goeckeler and Wysolmerski, 1995), suggesting that myosin II phosphorylation plays some important roles in the contraction and the motility in vivo. During the cell migration, the actin-based structures, called lamellipodia and filopodia, protrude at...