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An astral simulacrum of the central spindle accounts for normal, spindle-less, and anucleate cytokinesis in echinoderm embryos.

Su KC, Bement WM, Petronczki M, von Dassow G - Mol. Biol. Cell (2014)

Bottom Line: 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.We conclude that if multiple signals contribute to furrow induction in echinoderm embryos, they likely converge on the same signaling ensemble on an analogous cytoskeletal scaffold.

View Article: PubMed Central - PubMed

Affiliation: Oregon Institute of Marine Biology, Charleston, OR 97420 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, United Kingdom.

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Ect2 shapes the zone of active Rho. (A) Comparison of sand dollar embryos at the 16- to 32-cell division, expressing GFP-rGBD alone or with wt Ect2; projections of 15 (control) or 20 (Ect2) 1-μm sections (Supplemental Video S8). The dose of Ect2 used here was low enough that no overt defects were evident, but the embryo displays a broadened, brightened Rho zone. (B) Single surface section of 32-cell sand dollar embryo expressing GFP-rGBD and a dose of SpEct2 GEF4A at which no defects were apparent. False coloring was applied to highlight faint active Rho zones, which are not detectable in a projection of 11 1-μm sections displayed in the inset. (C) Representative projections of single sand dollar cells at equivalent cell sizes at onset of furrowing, injected with GFP-rGBD alone, or with low dose of wt Ect2 and GEF1A or GEF4A mutants. (D) Plot of cell size vs. width of Rho zone from images as in C. (E) Projection of all 28 (top) or four medial (lower) 0.8-μm sections of a dividing four-cell sand dollar embryo expressing eGFP-rGBD. Top right cell was also injected with mRNA encoding hyperactive SpEct2-CT and displays strong Rho activity over the entire surface.
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Figure 5: Ect2 shapes the zone of active Rho. (A) Comparison of sand dollar embryos at the 16- to 32-cell division, expressing GFP-rGBD alone or with wt Ect2; projections of 15 (control) or 20 (Ect2) 1-μm sections (Supplemental Video S8). The dose of Ect2 used here was low enough that no overt defects were evident, but the embryo displays a broadened, brightened Rho zone. (B) Single surface section of 32-cell sand dollar embryo expressing GFP-rGBD and a dose of SpEct2 GEF4A at which no defects were apparent. False coloring was applied to highlight faint active Rho zones, which are not detectable in a projection of 11 1-μm sections displayed in the inset. (C) Representative projections of single sand dollar cells at equivalent cell sizes at onset of furrowing, injected with GFP-rGBD alone, or with low dose of wt Ect2 and GEF1A or GEF4A mutants. (D) Plot of cell size vs. width of Rho zone from images as in C. (E) Projection of all 28 (top) or four medial (lower) 0.8-μm sections of a dividing four-cell sand dollar embryo expressing eGFP-rGBD. Top right cell was also injected with mRNA encoding hyperactive SpEct2-CT and displays strong Rho activity over the entire surface.

Mentions: Overexpression of wild-type Ect2 in urchins induces ectopic contraction, blebbing, and spindle slippage during furrow ingression (Figure 2), whereas expression of high levels of GEF-dead Ect2 GEF4A inhibits furrowing and cortical contraction, to the point of blocking cytokinesis completely (Figure 3). Because Rho is presumed to be the primary target of Ect2 during cytokinesis, we coexpressed wt or mutant Ect2 with eGFP–rhotekin GTPase–binding domain (rGBD; which detects active Rho specifically) to evaluate the effect of Ect2 on the spatial domain of Rho activation. In these experiments, we were careful to remain within a dosage range for Ect2 that was free of overt cytokinetic defects (such as the blebbing shown in Figure 2B) and at which visible Ect2 is confined to the equator during anaphase. Wt Ect2 induced a dramatically broader and brighter domain of Rho activity (Figure 5A and Supplemental Video S8). Conversely, expression of Ect2 GEF4A reduced the zone of active Rho to the point that it was barely detectable (Figure 5B). These effects were consistent across a range of mRNA dose and cell size (Figure 5, C and D). A milder hypomorphic single mutation of the GEF domain (V560A), which, based on results in other organisms, is expected to significantly reduce but not eliminate GEF activity (Liu et al., 1998; Propopenko et al., 1999; Schumacher et al., 2004), had no measurable effect on the cytokinetic Rho zone at equivalent expression levels. Increasing levels of wt Ect2 (i.e., to the range shown in Figure 2, A and B) also led to ectopic Rho activity outside the furrow zone, whereas excess Ect2 GEF4A prevented furrowing entirely. From these results, we conclude that the spatial pattern of Rho activation at the cell surface is determined directly by the localization and activity of Ect2.


An astral simulacrum of the central spindle accounts for normal, spindle-less, and anucleate cytokinesis in echinoderm embryos.

Su KC, Bement WM, Petronczki M, von Dassow G - Mol. Biol. Cell (2014)

Ect2 shapes the zone of active Rho. (A) Comparison of sand dollar embryos at the 16- to 32-cell division, expressing GFP-rGBD alone or with wt Ect2; projections of 15 (control) or 20 (Ect2) 1-μm sections (Supplemental Video S8). The dose of Ect2 used here was low enough that no overt defects were evident, but the embryo displays a broadened, brightened Rho zone. (B) Single surface section of 32-cell sand dollar embryo expressing GFP-rGBD and a dose of SpEct2 GEF4A at which no defects were apparent. False coloring was applied to highlight faint active Rho zones, which are not detectable in a projection of 11 1-μm sections displayed in the inset. (C) Representative projections of single sand dollar cells at equivalent cell sizes at onset of furrowing, injected with GFP-rGBD alone, or with low dose of wt Ect2 and GEF1A or GEF4A mutants. (D) Plot of cell size vs. width of Rho zone from images as in C. (E) Projection of all 28 (top) or four medial (lower) 0.8-μm sections of a dividing four-cell sand dollar embryo expressing eGFP-rGBD. Top right cell was also injected with mRNA encoding hyperactive SpEct2-CT and displays strong Rho activity over the entire surface.
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Related In: Results  -  Collection

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Figure 5: Ect2 shapes the zone of active Rho. (A) Comparison of sand dollar embryos at the 16- to 32-cell division, expressing GFP-rGBD alone or with wt Ect2; projections of 15 (control) or 20 (Ect2) 1-μm sections (Supplemental Video S8). The dose of Ect2 used here was low enough that no overt defects were evident, but the embryo displays a broadened, brightened Rho zone. (B) Single surface section of 32-cell sand dollar embryo expressing GFP-rGBD and a dose of SpEct2 GEF4A at which no defects were apparent. False coloring was applied to highlight faint active Rho zones, which are not detectable in a projection of 11 1-μm sections displayed in the inset. (C) Representative projections of single sand dollar cells at equivalent cell sizes at onset of furrowing, injected with GFP-rGBD alone, or with low dose of wt Ect2 and GEF1A or GEF4A mutants. (D) Plot of cell size vs. width of Rho zone from images as in C. (E) Projection of all 28 (top) or four medial (lower) 0.8-μm sections of a dividing four-cell sand dollar embryo expressing eGFP-rGBD. Top right cell was also injected with mRNA encoding hyperactive SpEct2-CT and displays strong Rho activity over the entire surface.
Mentions: Overexpression of wild-type Ect2 in urchins induces ectopic contraction, blebbing, and spindle slippage during furrow ingression (Figure 2), whereas expression of high levels of GEF-dead Ect2 GEF4A inhibits furrowing and cortical contraction, to the point of blocking cytokinesis completely (Figure 3). Because Rho is presumed to be the primary target of Ect2 during cytokinesis, we coexpressed wt or mutant Ect2 with eGFP–rhotekin GTPase–binding domain (rGBD; which detects active Rho specifically) to evaluate the effect of Ect2 on the spatial domain of Rho activation. In these experiments, we were careful to remain within a dosage range for Ect2 that was free of overt cytokinetic defects (such as the blebbing shown in Figure 2B) and at which visible Ect2 is confined to the equator during anaphase. Wt Ect2 induced a dramatically broader and brighter domain of Rho activity (Figure 5A and Supplemental Video S8). Conversely, expression of Ect2 GEF4A reduced the zone of active Rho to the point that it was barely detectable (Figure 5B). These effects were consistent across a range of mRNA dose and cell size (Figure 5, C and D). A milder hypomorphic single mutation of the GEF domain (V560A), which, based on results in other organisms, is expected to significantly reduce but not eliminate GEF activity (Liu et al., 1998; Propopenko et al., 1999; Schumacher et al., 2004), had no measurable effect on the cytokinetic Rho zone at equivalent expression levels. Increasing levels of wt Ect2 (i.e., to the range shown in Figure 2, A and B) also led to ectopic Rho activity outside the furrow zone, whereas excess Ect2 GEF4A prevented furrowing entirely. From these results, we conclude that the spatial pattern of Rho activation at the cell surface is determined directly by the localization and activity of Ect2.

Bottom Line: 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.We conclude that if multiple signals contribute to furrow induction in echinoderm embryos, they likely converge on the same signaling ensemble on an analogous cytoskeletal scaffold.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus