PgmNr Z6062: Cytoskeletal regulation by racgap1: required for more than just cytokinesis.

Authors:
S. E. Richards; R. M. Warga; D. A. Kane


Institutes
Western Michigan University, Kalamazoo, MI.


Abstract:

Cytokinesis is tightly regulated by the actions of a key protein complex known as centralspindlin. Composed of two dimers of mitotic kinesin-like protein MLKP1 and the Rho family GTPase-activating protein RacGAP1, centralspindlin positions and assembles the actomyosin ring driving furrow ingression and coordinates abscission. Like in files and worms, cells in the zebrafish racgap1 mutant, ogre (ogr), become binucleate and even quadnucleate during early development. Although cytokinesis fails, development proceeds albeit somewhat abnormally. Here we present evidence that RacGAP1 plays significant roles in other cellular processes independent of cytokinesis during development. Using confocal microscopy, we show that cell junctions in the mutant are disorganized; further inspection of the membrane reveals that β-catenin is no longer sequestered at the plasma membrane and instead is mislocalized to the cytoplasm. These results suggest that racgap1 may have a function in cell adhesion. To investigate this hypothesis further, we created double mutants with half baked (hab), carrying a mutation in the cell adhesion protein E-cadherin. Previously, we have shown that hab mutants have a zygotic maternal dominant effect that is expressed when both zygotic and maternal genomes are heterozygous for the mutant locus. These hab(-/+) embryos display an epiboly phenotype that is less severe than that of its homozygous mutant siblings; unlike the homozygous mutants, the heterozygous population is able to recover. Embryos that are ogr(-/-);hab(-/+) show a delay in epiboly that is more marked than hab(-/+) heterozygotes alone; interestingly, these double mutants display a distinctive, repetitive start and stop pattern that is unlike either the heterozygous or homozygous hab mutant. Supporting the idea that like E-cadherin, RacGAP1 regulates cell behavior by maintaining cell adhesion, we analyzed the collective migration of the dorsal forerunner cells (dfc) and found that ogr(-/-);hab(-/+) embryos have a greater tendency than hab(-/+) embryos for the dfc to break apart into three or more clusters. Therefore cells appear to be less adhesive when both racgap1 and E-cadherin are compromised. Our results also indicate that RacGAP1 may have a role in regulating the cytoskeletal framework surrounding the nucleus for when ogr(-/-);hab(-/+) embryos eventually reach somatogenesis, they develop a unique nuclear morphology. We conclude that the effects on cytokinesis have obscured the pleiotropic roles of RacGAP1 in mediating other cellular processes and that in particular it may be required for regulating the cytoskeleton at the cell surface and possibly at the nuclear membrane.



ZFIN Genetics Index
1. racgap1
2. cdh1