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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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Urchin Ect2 recruits to the central spindle and the astral microtubule ladder. (A) Schematic of urchin vs. human Ect2, showing positions of tags and derivatives used. (B) Wild-type 3xGFP Ect2 (gray) in 32-cell sand dollar coexpressed with mCh-H2B (red); single optical sections (Supplemental Video S1). (B′) Two-times magnification of the indicated cell immediately before, early in, and mid cleavage. (C) 3xGFP Ect2 GEF4A (gold) in 16-cell sand dollar embryo coexpressed with 2xmCh EMTB (cyan; Supplemental Video S2). (C′) Two-times magnification of the middle cell during the period when the astral ladder develops between central spindle and equatorial cortex.
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Figure 1: Urchin Ect2 recruits to the central spindle and the astral microtubule ladder. (A) Schematic of urchin vs. human Ect2, showing positions of tags and derivatives used. (B) Wild-type 3xGFP Ect2 (gray) in 32-cell sand dollar coexpressed with mCh-H2B (red); single optical sections (Supplemental Video S1). (B′) Two-times magnification of the indicated cell immediately before, early in, and mid cleavage. (C) 3xGFP Ect2 GEF4A (gold) in 16-cell sand dollar embryo coexpressed with 2xmCh EMTB (cyan; Supplemental Video S2). (C′) Two-times magnification of the middle cell during the period when the astral ladder develops between central spindle and equatorial cortex.

Mentions: To visualize Ect2 in early embryos, we fused purple urchin Ect2 (SpEct2) to 3x enhanced green fluorescent protein (eGFP; Figure 1A). When injected as mRNA into urchin or sand dollar zygotes at levels typically used for other probes, wild-type Ect2 caused overt defects as early as first cleavage (see later discussion). At much lower doses, however, embryos developed normally, and we could detect changing localization of Ect2 throughout the cell cycle as early as the eight-cell stage (Figure 1B, Supplemental Figure S1A, and Supplemental Video S1). As in other animal cells, Ect2 was nuclear during interphase, dispersed cytoplasmically in metaphase, and appeared on the central spindle just after anaphase onset. As anaphase progressed, we observed faint signal in the cytoplasmic disk between the central spindle and the equatorial cortex (Figure 1B and Supplemental Video S1). Although Ect2 did associate with the cell cortex in embryos during anaphase, especially when expressed at high levels, and we occasionally detected cortical enrichment in incipient furrows (Figure 1B′), we did not consistently observe a gradient of Ect2 at the equatorial membrane, as described in mammalian cells, in early embryos. By the blastula stage, however, once cells achieved the same scale as typical cultured somatic cells, we frequently observed equatorial cortical enrichment similar to mammalian cells (Supplemental Figure S1B). This distinction may, in principle, reflect detectability rather than actual presence or absence of an Ect2 gradient in the equatorial cortex. However, early embryonic cells that did exhibit clear cortical Ect2 also tended to exhibit abnormal behavior (described later), suggesting that the apparent difference between small and large cells is real: large cells lack the cortical gradient; midzone and the aster overlap region account for the bulk of localized 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)

Urchin Ect2 recruits to the central spindle and the astral microtubule ladder. (A) Schematic of urchin vs. human Ect2, showing positions of tags and derivatives used. (B) Wild-type 3xGFP Ect2 (gray) in 32-cell sand dollar coexpressed with mCh-H2B (red); single optical sections (Supplemental Video S1). (B′) Two-times magnification of the indicated cell immediately before, early in, and mid cleavage. (C) 3xGFP Ect2 GEF4A (gold) in 16-cell sand dollar embryo coexpressed with 2xmCh EMTB (cyan; Supplemental Video S2). (C′) Two-times magnification of the middle cell during the period when the astral ladder develops between central spindle and equatorial cortex.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263448&req=5

Figure 1: Urchin Ect2 recruits to the central spindle and the astral microtubule ladder. (A) Schematic of urchin vs. human Ect2, showing positions of tags and derivatives used. (B) Wild-type 3xGFP Ect2 (gray) in 32-cell sand dollar coexpressed with mCh-H2B (red); single optical sections (Supplemental Video S1). (B′) Two-times magnification of the indicated cell immediately before, early in, and mid cleavage. (C) 3xGFP Ect2 GEF4A (gold) in 16-cell sand dollar embryo coexpressed with 2xmCh EMTB (cyan; Supplemental Video S2). (C′) Two-times magnification of the middle cell during the period when the astral ladder develops between central spindle and equatorial cortex.
Mentions: To visualize Ect2 in early embryos, we fused purple urchin Ect2 (SpEct2) to 3x enhanced green fluorescent protein (eGFP; Figure 1A). When injected as mRNA into urchin or sand dollar zygotes at levels typically used for other probes, wild-type Ect2 caused overt defects as early as first cleavage (see later discussion). At much lower doses, however, embryos developed normally, and we could detect changing localization of Ect2 throughout the cell cycle as early as the eight-cell stage (Figure 1B, Supplemental Figure S1A, and Supplemental Video S1). As in other animal cells, Ect2 was nuclear during interphase, dispersed cytoplasmically in metaphase, and appeared on the central spindle just after anaphase onset. As anaphase progressed, we observed faint signal in the cytoplasmic disk between the central spindle and the equatorial cortex (Figure 1B and Supplemental Video S1). Although Ect2 did associate with the cell cortex in embryos during anaphase, especially when expressed at high levels, and we occasionally detected cortical enrichment in incipient furrows (Figure 1B′), we did not consistently observe a gradient of Ect2 at the equatorial membrane, as described in mammalian cells, in early embryos. By the blastula stage, however, once cells achieved the same scale as typical cultured somatic cells, we frequently observed equatorial cortical enrichment similar to mammalian cells (Supplemental Figure S1B). This distinction may, in principle, reflect detectability rather than actual presence or absence of an Ect2 gradient in the equatorial cortex. However, early embryonic cells that did exhibit clear cortical Ect2 also tended to exhibit abnormal behavior (described later), suggesting that the apparent difference between small and large cells is real: large cells lack the cortical gradient; midzone and the aster overlap region account for the bulk of localized 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