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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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Physical displacement of the cortex relative to the spindle midplane modifies the RhoA zone. All times in this figure are in minutes:seconds relative to the physical perturbation indicated in the diagrams on the left. In each case, the dashed outlines in the first frame show the position of the spindle and the glass needle. Blastomeres from green urchin embryos at the 32–64-cell stage were used for these experiments. (A) Projection of four sections through a cell upon which the needle was pressed slightly to one side of the spindle, distending a portion of the cell into a pouch ∼10 μm thick. The side nearer the spindle initiates a tightly focused RhoA activity-rich furrow that ingresses (arrows); the far side accumulates active RhoA (brackets) but fails to focus it, and a series of shallow, unstable furrows develop and regress. (B) Projection of nine sections through a blastomere in which the spindle was displaced along the polar axis after furrow initiation. In the first frame, the blunt-ended needle is parked against one pole of the cell, slightly denting it. At 00:00, the needle was advanced by ∼15 μm (without penetrating the cell membrane), shoving the spindle so that the midplane shifts up relative to the furrow. Arrow and bracket in 00:20 mark the position immediately after the shove of the furrow and the RhoA zone, respectively, and remain the same for comparison in subsequent frames. The RhoA zone climbs higher on the cortex after the spindle midplane and is followed, in turn, by the furrow (second set of brackets and arrows in 03:00–06:40) such that the cell cleaves asymmetrically. (C) Projection of eight sections through a cell subjected to the converse of B: the cortex was displaced by a blunt needle toward the spindle midzone. In −00:20, the needle is parked slightly off center; at 00:00, the needle was advanced to bring a patch of nonequatorial cortex deep into the equatorial zone. At 06:20, needle tip shows accumulation of active RhoA (arrow). In addition, the original RhoA zone slides up the cortex (brackets in 04:00–08:40), apparently after half of the mitotic apparatus is broken by the needle. See online supplemental material for videos corresponding to A (Video 8) and B (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.
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fig6: Physical displacement of the cortex relative to the spindle midplane modifies the RhoA zone. All times in this figure are in minutes:seconds relative to the physical perturbation indicated in the diagrams on the left. In each case, the dashed outlines in the first frame show the position of the spindle and the glass needle. Blastomeres from green urchin embryos at the 32–64-cell stage were used for these experiments. (A) Projection of four sections through a cell upon which the needle was pressed slightly to one side of the spindle, distending a portion of the cell into a pouch ∼10 μm thick. The side nearer the spindle initiates a tightly focused RhoA activity-rich furrow that ingresses (arrows); the far side accumulates active RhoA (brackets) but fails to focus it, and a series of shallow, unstable furrows develop and regress. (B) Projection of nine sections through a blastomere in which the spindle was displaced along the polar axis after furrow initiation. In the first frame, the blunt-ended needle is parked against one pole of the cell, slightly denting it. At 00:00, the needle was advanced by ∼15 μm (without penetrating the cell membrane), shoving the spindle so that the midplane shifts up relative to the furrow. Arrow and bracket in 00:20 mark the position immediately after the shove of the furrow and the RhoA zone, respectively, and remain the same for comparison in subsequent frames. The RhoA zone climbs higher on the cortex after the spindle midplane and is followed, in turn, by the furrow (second set of brackets and arrows in 03:00–06:40) such that the cell cleaves asymmetrically. (C) Projection of eight sections through a cell subjected to the converse of B: the cortex was displaced by a blunt needle toward the spindle midzone. In −00:20, the needle is parked slightly off center; at 00:00, the needle was advanced to bring a patch of nonequatorial cortex deep into the equatorial zone. At 06:20, needle tip shows accumulation of active RhoA (arrow). In addition, the original RhoA zone slides up the cortex (brackets in 04:00–08:40), apparently after half of the mitotic apparatus is broken by the needle. See online supplemental material for videos corresponding to A (Video 8) and B (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.

Mentions: We first tested whether isolating the cortex from the spindle would prevent formation of the zone. We flattened cells with a microneedle (Fig. 6 A and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1) so as to impose the spindle on one region of the cortex while isolating the opposite edge in a flattened pouch. This manipulation resulted in a unilateral furrow and a focused RhoA zone that formed on the side of the cell closest to the displaced spindle (Rappaport and Conrad, 1963). In contrast, on the side distal to the displaced spindle, a low level of active RhoA was distributed along the entire cell edge and failed to focus into a discrete zone. No furrow formed on this side of the cell, and the RhoA-bright furrow on the spindle side divided the cell unilaterally. Thus, spindle displacement results in a corresponding change in the RhoA activity zone, which is consistent with the zone directing cytokinetic apparatus assembly.


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

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

Physical displacement of the cortex relative to the spindle midplane modifies the RhoA zone. All times in this figure are in minutes:seconds relative to the physical perturbation indicated in the diagrams on the left. In each case, the dashed outlines in the first frame show the position of the spindle and the glass needle. Blastomeres from green urchin embryos at the 32–64-cell stage were used for these experiments. (A) Projection of four sections through a cell upon which the needle was pressed slightly to one side of the spindle, distending a portion of the cell into a pouch ∼10 μm thick. The side nearer the spindle initiates a tightly focused RhoA activity-rich furrow that ingresses (arrows); the far side accumulates active RhoA (brackets) but fails to focus it, and a series of shallow, unstable furrows develop and regress. (B) Projection of nine sections through a blastomere in which the spindle was displaced along the polar axis after furrow initiation. In the first frame, the blunt-ended needle is parked against one pole of the cell, slightly denting it. At 00:00, the needle was advanced by ∼15 μm (without penetrating the cell membrane), shoving the spindle so that the midplane shifts up relative to the furrow. Arrow and bracket in 00:20 mark the position immediately after the shove of the furrow and the RhoA zone, respectively, and remain the same for comparison in subsequent frames. The RhoA zone climbs higher on the cortex after the spindle midplane and is followed, in turn, by the furrow (second set of brackets and arrows in 03:00–06:40) such that the cell cleaves asymmetrically. (C) Projection of eight sections through a cell subjected to the converse of B: the cortex was displaced by a blunt needle toward the spindle midzone. In −00:20, the needle is parked slightly off center; at 00:00, the needle was advanced to bring a patch of nonequatorial cortex deep into the equatorial zone. At 06:20, needle tip shows accumulation of active RhoA (arrow). In addition, the original RhoA zone slides up the cortex (brackets in 04:00–08:40), apparently after half of the mitotic apparatus is broken by the needle. See online supplemental material for videos corresponding to A (Video 8) and B (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.
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fig6: Physical displacement of the cortex relative to the spindle midplane modifies the RhoA zone. All times in this figure are in minutes:seconds relative to the physical perturbation indicated in the diagrams on the left. In each case, the dashed outlines in the first frame show the position of the spindle and the glass needle. Blastomeres from green urchin embryos at the 32–64-cell stage were used for these experiments. (A) Projection of four sections through a cell upon which the needle was pressed slightly to one side of the spindle, distending a portion of the cell into a pouch ∼10 μm thick. The side nearer the spindle initiates a tightly focused RhoA activity-rich furrow that ingresses (arrows); the far side accumulates active RhoA (brackets) but fails to focus it, and a series of shallow, unstable furrows develop and regress. (B) Projection of nine sections through a blastomere in which the spindle was displaced along the polar axis after furrow initiation. In the first frame, the blunt-ended needle is parked against one pole of the cell, slightly denting it. At 00:00, the needle was advanced by ∼15 μm (without penetrating the cell membrane), shoving the spindle so that the midplane shifts up relative to the furrow. Arrow and bracket in 00:20 mark the position immediately after the shove of the furrow and the RhoA zone, respectively, and remain the same for comparison in subsequent frames. The RhoA zone climbs higher on the cortex after the spindle midplane and is followed, in turn, by the furrow (second set of brackets and arrows in 03:00–06:40) such that the cell cleaves asymmetrically. (C) Projection of eight sections through a cell subjected to the converse of B: the cortex was displaced by a blunt needle toward the spindle midzone. In −00:20, the needle is parked slightly off center; at 00:00, the needle was advanced to bring a patch of nonequatorial cortex deep into the equatorial zone. At 06:20, needle tip shows accumulation of active RhoA (arrow). In addition, the original RhoA zone slides up the cortex (brackets in 04:00–08:40), apparently after half of the mitotic apparatus is broken by the needle. See online supplemental material for videos corresponding to A (Video 8) and B (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.
Mentions: We first tested whether isolating the cortex from the spindle would prevent formation of the zone. We flattened cells with a microneedle (Fig. 6 A and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1) so as to impose the spindle on one region of the cortex while isolating the opposite edge in a flattened pouch. This manipulation resulted in a unilateral furrow and a focused RhoA zone that formed on the side of the cell closest to the displaced spindle (Rappaport and Conrad, 1963). In contrast, on the side distal to the displaced spindle, a low level of active RhoA was distributed along the entire cell edge and failed to focus into a discrete zone. No furrow formed on this side of the cell, and the RhoA-bright furrow on the spindle side divided the cell unilaterally. Thus, spindle displacement results in a corresponding change in the RhoA activity zone, which is consistent with the zone directing cytokinetic apparatus assembly.

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