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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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Microtubule disruption disrupts RhoA zone. (A) Projection of seven sections through a purple urchin embryo, which was first treated with 12 μM cytochalasin D immediately before furrowing, and then treated with 25 μM nocodazole at the time the furrows began to regress (00:00). Nocodazole causes rapid abolition of RhoA zones (arrowheads). Bar, 25 μm. (B) Projection of 18 sections through purple urchin embryo cultured in 5 nM nocodazole for 30 min before filming. Furrows in top two cells are associated with uneven, poorly bounded zones of RhoA activity (arrowheads, 00:00). Zones subsequently fragment, and furrows regress. (C) Projection of 18 sections through a control purple urchin embryo showing normal width and brightness of the RhoA zone during telophase. (D) Projection of 18 sections through a four-cell purple urchin embryo cultured in 5 nM nocodazole. The RhoA zone is much wider than controls (compare brackets in C and D). (E) Scatter plot showing that RhoA zones are consistently wider in embryos treated with 5 nM nocodazole. Zones were measured at the point just after ingression begins (exemplified by the lower left furrows in C and D) in four untreated and six treated embryos of equivalent developmental stage (four and eight cell) from identical experiments on two successive days. Measurements from the same embryo are aligned vertically. Control and nocodazole-treated cells are significantly different (t test: P = 10−6).
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fig5: Microtubule disruption disrupts RhoA zone. (A) Projection of seven sections through a purple urchin embryo, which was first treated with 12 μM cytochalasin D immediately before furrowing, and then treated with 25 μM nocodazole at the time the furrows began to regress (00:00). Nocodazole causes rapid abolition of RhoA zones (arrowheads). Bar, 25 μm. (B) Projection of 18 sections through purple urchin embryo cultured in 5 nM nocodazole for 30 min before filming. Furrows in top two cells are associated with uneven, poorly bounded zones of RhoA activity (arrowheads, 00:00). Zones subsequently fragment, and furrows regress. (C) Projection of 18 sections through a control purple urchin embryo showing normal width and brightness of the RhoA zone during telophase. (D) Projection of 18 sections through a four-cell purple urchin embryo cultured in 5 nM nocodazole. The RhoA zone is much wider than controls (compare brackets in C and D). (E) Scatter plot showing that RhoA zones are consistently wider in embryos treated with 5 nM nocodazole. Zones were measured at the point just after ingression begins (exemplified by the lower left furrows in C and D) in four untreated and six treated embryos of equivalent developmental stage (four and eight cell) from identical experiments on two successive days. Measurements from the same embryo are aligned vertically. Control and nocodazole-treated cells are significantly different (t test: P = 10−6).

Mentions: Curiously, active RhoA was often observed to dart away from the equatorial zone inward on linear tracks that extended toward the spindle poles (unpublished data; Fig. 4 F). The inward extension of linear tracks that are rich in active RhoA could be seen in single optical sections and were particularly striking in green urchins (Fig. 4 E and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1), allowing quantification of the rate of inward movement (0.1–0.5 μm/s; mean = 0.2 μm/s; n = 116). These objects were not confined to the equator of cytochalasin-treated cells but were consistently brighter in the region where the furrow would have formed. Three-dimensional projections from single time points (Fig. 4 F) and brightest point projections from multiple time points (unpublished data) showed clearly that the linear tracks point toward the spindle poles. The formation of linear extensions was nocodazole sensitive (see Fig. 5 A), indicating that they are microtubule dependent.


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

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

Microtubule disruption disrupts RhoA zone. (A) Projection of seven sections through a purple urchin embryo, which was first treated with 12 μM cytochalasin D immediately before furrowing, and then treated with 25 μM nocodazole at the time the furrows began to regress (00:00). Nocodazole causes rapid abolition of RhoA zones (arrowheads). Bar, 25 μm. (B) Projection of 18 sections through purple urchin embryo cultured in 5 nM nocodazole for 30 min before filming. Furrows in top two cells are associated with uneven, poorly bounded zones of RhoA activity (arrowheads, 00:00). Zones subsequently fragment, and furrows regress. (C) Projection of 18 sections through a control purple urchin embryo showing normal width and brightness of the RhoA zone during telophase. (D) Projection of 18 sections through a four-cell purple urchin embryo cultured in 5 nM nocodazole. The RhoA zone is much wider than controls (compare brackets in C and D). (E) Scatter plot showing that RhoA zones are consistently wider in embryos treated with 5 nM nocodazole. Zones were measured at the point just after ingression begins (exemplified by the lower left furrows in C and D) in four untreated and six treated embryos of equivalent developmental stage (four and eight cell) from identical experiments on two successive days. Measurements from the same embryo are aligned vertically. Control and nocodazole-treated cells are significantly different (t test: P = 10−6).
© Copyright Policy
Related In: Results  -  Collection

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fig5: Microtubule disruption disrupts RhoA zone. (A) Projection of seven sections through a purple urchin embryo, which was first treated with 12 μM cytochalasin D immediately before furrowing, and then treated with 25 μM nocodazole at the time the furrows began to regress (00:00). Nocodazole causes rapid abolition of RhoA zones (arrowheads). Bar, 25 μm. (B) Projection of 18 sections through purple urchin embryo cultured in 5 nM nocodazole for 30 min before filming. Furrows in top two cells are associated with uneven, poorly bounded zones of RhoA activity (arrowheads, 00:00). Zones subsequently fragment, and furrows regress. (C) Projection of 18 sections through a control purple urchin embryo showing normal width and brightness of the RhoA zone during telophase. (D) Projection of 18 sections through a four-cell purple urchin embryo cultured in 5 nM nocodazole. The RhoA zone is much wider than controls (compare brackets in C and D). (E) Scatter plot showing that RhoA zones are consistently wider in embryos treated with 5 nM nocodazole. Zones were measured at the point just after ingression begins (exemplified by the lower left furrows in C and D) in four untreated and six treated embryos of equivalent developmental stage (four and eight cell) from identical experiments on two successive days. Measurements from the same embryo are aligned vertically. Control and nocodazole-treated cells are significantly different (t test: P = 10−6).
Mentions: Curiously, active RhoA was often observed to dart away from the equatorial zone inward on linear tracks that extended toward the spindle poles (unpublished data; Fig. 4 F). The inward extension of linear tracks that are rich in active RhoA could be seen in single optical sections and were particularly striking in green urchins (Fig. 4 E and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1), allowing quantification of the rate of inward movement (0.1–0.5 μm/s; mean = 0.2 μm/s; n = 116). These objects were not confined to the equator of cytochalasin-treated cells but were consistently brighter in the region where the furrow would have formed. Three-dimensional projections from single time points (Fig. 4 F) and brightest point projections from multiple time points (unpublished data) showed clearly that the linear tracks point toward the spindle poles. The formation of linear extensions was nocodazole sensitive (see Fig. 5 A), indicating that they are microtubule dependent.

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