An astral simulacrum of the central spindle accounts for normal, spindle-less, and anucleate cytokinesis in echinoderm embryos.
Bottom Line: Although the nature, origin, and variety of such cues have long been obscure, one component is certainly the Rho activator Ect2.Here we describe the behavior and function of Ect2 in echinoderm embryos, showing that Ect2 migrates from spindle midzone to astral microtubules in anaphase and that Ect2 shapes the pattern of Rho activation in incipient furrows.In all these cases, the cell assembles essentially the same cytokinetic signaling ensemble—opposed astral microtubules decorated with Ect2 and Cyk4.
Affiliation: Oregon Institute of Marine Biology, Charleston, OR 97420 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, United Kingdom.Show MeSH
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Mentions: The torus experiment is a somewhat more rigorous test than a geometrically simple cell with parallel spindles, for example, which arise after a cytokinesis failure (Salmon and Wolniak, 1990; Argiros et al., 2012), because the torus experiment excludes the possibility that spatial information derived from the previously failed cytokinesis may remain to contribute, positively or negatively, to specification of the secondary furrow. It also physically occludes the primary from the secondary zone, whereas these are continuous in a geometrically simple binucleate cell. However, in both cases, the cell contains spindles, chromosomes, and, as events proceed, nuclei. We therefore created anucleate, centrosome-containing cytoplasts from sand dollar or starfish embryos expressing either labeled wt or mutant Ect2 or Cyk4. This was accomplished by cutting metaphase cells with a glass needle passing between one pole and the rest of the mitotic apparatus. We used labeled histones or Hoechst 33342 to verify the absence of chromosomes from cytoplasts. After a small delay, the centrosome-containing cytoplast begins cyclical centrosome duplication. Although cytoplasts made from normal cells generate furrows at respectable frequency (von Dassow et al., 2009), we found it impossible, despite numerous attempts, to achieve reliable furrowing from cytoplasts expressing excess wt or mutant Ect2. In the absence of a spindle or nuclei, exogenous wt Ect2 associated with the cortex and caused excessive contractility, whereas GEF-defective mutants strongly inhibited furrowing by cytoplasts. In a few out of very many cases, we detected wt or GEF4A Ect2 faintly in the overlap region between asters or associated with ingressing furrows (unpublished data). Cytoplasts from starfish embryos expressing exogenous Cyk4 had no such difficulties, however, and cleaved to completion consistently. Cyk4 unambiguously recruited to overlapping astral microtubules between pairs of centrosomes, concentrated beneath the “equatorial” cortex, and was gathered by the ingressing furrow into a midbody (Figure 8 and Supplemental Video S11). Cyk4-expressing cytoplasts from sand dollar embryos similarly concentrated Cyk4 into midbodies (unpublished data), although with far lower expression level.
Affiliation: Oregon Institute of Marine Biology, Charleston, OR 97420 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, United Kingdom.