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A microtubule-dependent zone of active RhoA during cleavage plane specification.

Bement WM, Benink HA, von Dassow G - J. Cell Biol. (2005)

Bottom Line: Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis.Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement.We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Dynamics, Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, USA. wmbement@wisc.edu

ABSTRACT
Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

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RhoA zones accompany divergent forms of cytokinesis. (A) Micromere formation in green urchin embryos. Projection of 12 1-μm sections of an eight-cell green urchin embryo. Dashed outlines in first frame show spindle position and orientation. Cell at top forms a circumferential furrow above the spindle midplane; in other cells, the asymmetric position of the spindle corresponds to asymmetric furrowing, and the region of concentrated eGFP-rGBD spreads around the circumference as furrowing proceeds. (B) Polar body formation in X. laevis oocytes. Projection of 13 optical sections showing that eGFP-rGBD becomes concentrated in a circular region at the animal pole and constricts inward. Arrowheads indicate edges of zone. (C) Dual-labelled images from projection of 13 optical planes showing tubulin (red) and eGFP-rGBD (green) during polar body emission. Top row shows facing view; circular region of concentrated eGFP-rGBD closes inward around microtubules of the first meiotic spindle. Bottom three rows show Z view in which eGFP-rGBD is seen to contract inward and downward in concert with the closure of the cytokinetic array, which pinches off the forming polar body. The microscope was refocused at 05:20 to allow the eGFP-rGBD ring to be followed deeper into the cytoplasm. See online supplemental material for videos corresponding to A (Video 4) and C (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). All times are in minutes:seconds after filming began. Bars, 25 μm.
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fig3: RhoA zones accompany divergent forms of cytokinesis. (A) Micromere formation in green urchin embryos. Projection of 12 1-μm sections of an eight-cell green urchin embryo. Dashed outlines in first frame show spindle position and orientation. Cell at top forms a circumferential furrow above the spindle midplane; in other cells, the asymmetric position of the spindle corresponds to asymmetric furrowing, and the region of concentrated eGFP-rGBD spreads around the circumference as furrowing proceeds. (B) Polar body formation in X. laevis oocytes. Projection of 13 optical sections showing that eGFP-rGBD becomes concentrated in a circular region at the animal pole and constricts inward. Arrowheads indicate edges of zone. (C) Dual-labelled images from projection of 13 optical planes showing tubulin (red) and eGFP-rGBD (green) during polar body emission. Top row shows facing view; circular region of concentrated eGFP-rGBD closes inward around microtubules of the first meiotic spindle. Bottom three rows show Z view in which eGFP-rGBD is seen to contract inward and downward in concert with the closure of the cytokinetic array, which pinches off the forming polar body. The microscope was refocused at 05:20 to allow the eGFP-rGBD ring to be followed deeper into the cytoplasm. See online supplemental material for videos corresponding to A (Video 4) and C (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). All times are in minutes:seconds after filming began. Bars, 25 μm.

Mentions: All of the previous results depict divisions in which the cleavage furrow forms circumferentially between two equivalent mitotic asters deep in the cell. We also investigated RhoA activity in two specialized forms of cytokinesis: micromere formation in sea urchins and polar body formation in X. laevis. At the eight-cell stage in sea urchins, the four vegetal cells divide highly asymmetrically such that the vegetal-most daughter is ∼1/30 the volume of the other. In preparation for micromere formation, the mitotic spindle develops in an eccentric position with one aster flattened against the vegetal cortex and the other deep in the cytoplasm. During micromere formation, the RhoA zone was asymmetrically positioned toward the inner edge of the blastomeres, and both formed and focused first on the side of the blastomere, where the spindle was closest, corresponding to the site of furrow initiation (Fig. 3 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1).


A microtubule-dependent zone of active RhoA during cleavage plane specification.

Bement WM, Benink HA, von Dassow G - J. Cell Biol. (2005)

RhoA zones accompany divergent forms of cytokinesis. (A) Micromere formation in green urchin embryos. Projection of 12 1-μm sections of an eight-cell green urchin embryo. Dashed outlines in first frame show spindle position and orientation. Cell at top forms a circumferential furrow above the spindle midplane; in other cells, the asymmetric position of the spindle corresponds to asymmetric furrowing, and the region of concentrated eGFP-rGBD spreads around the circumference as furrowing proceeds. (B) Polar body formation in X. laevis oocytes. Projection of 13 optical sections showing that eGFP-rGBD becomes concentrated in a circular region at the animal pole and constricts inward. Arrowheads indicate edges of zone. (C) Dual-labelled images from projection of 13 optical planes showing tubulin (red) and eGFP-rGBD (green) during polar body emission. Top row shows facing view; circular region of concentrated eGFP-rGBD closes inward around microtubules of the first meiotic spindle. Bottom three rows show Z view in which eGFP-rGBD is seen to contract inward and downward in concert with the closure of the cytokinetic array, which pinches off the forming polar body. The microscope was refocused at 05:20 to allow the eGFP-rGBD ring to be followed deeper into the cytoplasm. See online supplemental material for videos corresponding to A (Video 4) and C (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). All times are in minutes:seconds after filming began. Bars, 25 μm.
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fig3: RhoA zones accompany divergent forms of cytokinesis. (A) Micromere formation in green urchin embryos. Projection of 12 1-μm sections of an eight-cell green urchin embryo. Dashed outlines in first frame show spindle position and orientation. Cell at top forms a circumferential furrow above the spindle midplane; in other cells, the asymmetric position of the spindle corresponds to asymmetric furrowing, and the region of concentrated eGFP-rGBD spreads around the circumference as furrowing proceeds. (B) Polar body formation in X. laevis oocytes. Projection of 13 optical sections showing that eGFP-rGBD becomes concentrated in a circular region at the animal pole and constricts inward. Arrowheads indicate edges of zone. (C) Dual-labelled images from projection of 13 optical planes showing tubulin (red) and eGFP-rGBD (green) during polar body emission. Top row shows facing view; circular region of concentrated eGFP-rGBD closes inward around microtubules of the first meiotic spindle. Bottom three rows show Z view in which eGFP-rGBD is seen to contract inward and downward in concert with the closure of the cytokinetic array, which pinches off the forming polar body. The microscope was refocused at 05:20 to allow the eGFP-rGBD ring to be followed deeper into the cytoplasm. See online supplemental material for videos corresponding to A (Video 4) and C (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). All times are in minutes:seconds after filming began. Bars, 25 μm.
Mentions: All of the previous results depict divisions in which the cleavage furrow forms circumferentially between two equivalent mitotic asters deep in the cell. We also investigated RhoA activity in two specialized forms of cytokinesis: micromere formation in sea urchins and polar body formation in X. laevis. At the eight-cell stage in sea urchins, the four vegetal cells divide highly asymmetrically such that the vegetal-most daughter is ∼1/30 the volume of the other. In preparation for micromere formation, the mitotic spindle develops in an eccentric position with one aster flattened against the vegetal cortex and the other deep in the cytoplasm. During micromere formation, the RhoA zone was asymmetrically positioned toward the inner edge of the blastomeres, and both formed and focused first on the side of the blastomere, where the spindle was closest, corresponding to the site of furrow initiation (Fig. 3 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1).

Bottom Line: Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis.Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement.We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Dynamics, Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, USA. wmbement@wisc.edu

ABSTRACT
Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.

Show MeSH
Related in: MedlinePlus