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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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Each half of the mitotic apparatus can induce one zone of RhoA activation and a corresponding furrow. Times are in minutes:seconds after the onset of filming (which began within a minute after cutting); dashed outline indicates the position of the needle. (A) Projection of eight sections through a cell bisected during metaphase. Arrowheads in the first frame indicate the centrosomes, which soon disappear from view. At approximately the time this cell would have entered anaphase, one patch of cortex near the spindle half in each cell fragment accumulates eGFP-rGBD (arrows, 06:00). (B) Projection of 10 sections through a cell cut in anaphase. The left spindle half moved away from the cut, whereas the right spindle half remained closer to the cut edge. In the left half, active RhoA immediately accumulated on the cut face near the site where the cut passed through the spindle. Within minutes, however, the spindle half in the left fragment induced a new zone (arrows in second frame), which proceeded to ingress, forming a tripartite furrow. The right half underwent unequal cleavage in association with an asymmetric RhoA zone and furrow. See Video 10 (available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). (C) Projection of 12 sections through a cell cut (as a control for A and B) such that the entire spindle (dashed outline, 00:00) remains in one of the two fragments. Only the spindle-containing fragment exhibited elevated RhoA activity (arrow) and a cleavage furrow. Bars, 25 μm.
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fig7: Each half of the mitotic apparatus can induce one zone of RhoA activation and a corresponding furrow. Times are in minutes:seconds after the onset of filming (which began within a minute after cutting); dashed outline indicates the position of the needle. (A) Projection of eight sections through a cell bisected during metaphase. Arrowheads in the first frame indicate the centrosomes, which soon disappear from view. At approximately the time this cell would have entered anaphase, one patch of cortex near the spindle half in each cell fragment accumulates eGFP-rGBD (arrows, 06:00). (B) Projection of 10 sections through a cell cut in anaphase. The left spindle half moved away from the cut, whereas the right spindle half remained closer to the cut edge. In the left half, active RhoA immediately accumulated on the cut face near the site where the cut passed through the spindle. Within minutes, however, the spindle half in the left fragment induced a new zone (arrows in second frame), which proceeded to ingress, forming a tripartite furrow. The right half underwent unequal cleavage in association with an asymmetric RhoA zone and furrow. See Video 10 (available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). (C) Projection of 12 sections through a cell cut (as a control for A and B) such that the entire spindle (dashed outline, 00:00) remains in one of the two fragments. Only the spindle-containing fragment exhibited elevated RhoA activity (arrow) and a cleavage furrow. Bars, 25 μm.

Mentions: In normal cells, the furrow forms between two juxtaposed mitotic asters. However, recent results show that monastral spindles can induce cytokinetic furrowing (Canman et al., 2003; Alsop and Zhang, 2004). Therefore, we tested whether half spindles could induce localized RhoA activation by using a glass microneedle to bisect cells between the two asters in metaphase or anaphase. The needle was slowly lowered across the cell from one side to the other, taking special care to avoid any rupture to the cell membrane because RhoA is activated by cellular wounding (Benink and Bement, 2005). In four of five cells successfully bisected, both halves exhibited a localized elevation of RhoA activity associated with the half spindle (in the fifth case, zone formation may have occurred somewhere beyond the imaged region of the cell). Fig. 7 A depicts a cell bisected in metaphase. Starting a few minutes after cutting, RhoA activation was observed immediately adjacent to the cut spindle halves. In both halves, these zones faded minutes later without inducing a furrow. Fig. 7 B (Video 10, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1) depicts a cell bisected in anaphase. Immediately after the cut, the much larger anaphase half spindles rapidly induced bright RhoA activity zones that are associated with dramatic, and in some cases multiple, furrows. Fig. 7 C shows a control cell in which the bisection separated a pouch of cytoplasm without any spindle. In all of six such cases, the spindle-containing fragment cleaved with a normal RhoA zone when the bisection took place in metaphase or before, whereas the enucleate fragment never exhibited any elevated RhoA activity.


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

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

Each half of the mitotic apparatus can induce one zone of RhoA activation and a corresponding furrow. Times are in minutes:seconds after the onset of filming (which began within a minute after cutting); dashed outline indicates the position of the needle. (A) Projection of eight sections through a cell bisected during metaphase. Arrowheads in the first frame indicate the centrosomes, which soon disappear from view. At approximately the time this cell would have entered anaphase, one patch of cortex near the spindle half in each cell fragment accumulates eGFP-rGBD (arrows, 06:00). (B) Projection of 10 sections through a cell cut in anaphase. The left spindle half moved away from the cut, whereas the right spindle half remained closer to the cut edge. In the left half, active RhoA immediately accumulated on the cut face near the site where the cut passed through the spindle. Within minutes, however, the spindle half in the left fragment induced a new zone (arrows in second frame), which proceeded to ingress, forming a tripartite furrow. The right half underwent unequal cleavage in association with an asymmetric RhoA zone and furrow. See Video 10 (available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). (C) Projection of 12 sections through a cell cut (as a control for A and B) such that the entire spindle (dashed outline, 00:00) remains in one of the two fragments. Only the spindle-containing fragment exhibited elevated RhoA activity (arrow) and a cleavage furrow. Bars, 25 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171391&req=5

fig7: Each half of the mitotic apparatus can induce one zone of RhoA activation and a corresponding furrow. Times are in minutes:seconds after the onset of filming (which began within a minute after cutting); dashed outline indicates the position of the needle. (A) Projection of eight sections through a cell bisected during metaphase. Arrowheads in the first frame indicate the centrosomes, which soon disappear from view. At approximately the time this cell would have entered anaphase, one patch of cortex near the spindle half in each cell fragment accumulates eGFP-rGBD (arrows, 06:00). (B) Projection of 10 sections through a cell cut in anaphase. The left spindle half moved away from the cut, whereas the right spindle half remained closer to the cut edge. In the left half, active RhoA immediately accumulated on the cut face near the site where the cut passed through the spindle. Within minutes, however, the spindle half in the left fragment induced a new zone (arrows in second frame), which proceeded to ingress, forming a tripartite furrow. The right half underwent unequal cleavage in association with an asymmetric RhoA zone and furrow. See Video 10 (available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). (C) Projection of 12 sections through a cell cut (as a control for A and B) such that the entire spindle (dashed outline, 00:00) remains in one of the two fragments. Only the spindle-containing fragment exhibited elevated RhoA activity (arrow) and a cleavage furrow. Bars, 25 μm.
Mentions: In normal cells, the furrow forms between two juxtaposed mitotic asters. However, recent results show that monastral spindles can induce cytokinetic furrowing (Canman et al., 2003; Alsop and Zhang, 2004). Therefore, we tested whether half spindles could induce localized RhoA activation by using a glass microneedle to bisect cells between the two asters in metaphase or anaphase. The needle was slowly lowered across the cell from one side to the other, taking special care to avoid any rupture to the cell membrane because RhoA is activated by cellular wounding (Benink and Bement, 2005). In four of five cells successfully bisected, both halves exhibited a localized elevation of RhoA activity associated with the half spindle (in the fifth case, zone formation may have occurred somewhere beyond the imaged region of the cell). Fig. 7 A depicts a cell bisected in metaphase. Starting a few minutes after cutting, RhoA activation was observed immediately adjacent to the cut spindle halves. In both halves, these zones faded minutes later without inducing a furrow. Fig. 7 B (Video 10, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1) depicts a cell bisected in anaphase. Immediately after the cut, the much larger anaphase half spindles rapidly induced bright RhoA activity zones that are associated with dramatic, and in some cases multiple, furrows. Fig. 7 C shows a control cell in which the bisection separated a pouch of cytoplasm without any spindle. In all of six such cases, the spindle-containing fragment cleaved with a normal RhoA zone when the bisection took place in metaphase or before, whereas the enucleate fragment never exhibited any elevated RhoA activity.

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