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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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Characteristics of RhoA activity zones. (A) Single optical sections through a green urchin blastomere before, early, and late in furrowing showing zone in cross section. Bar, 25 μm. (B) Single optical sections through a blastomere from green urchin embryo showing eGFP-rGBD accumulation in spindle region. Chromosomes appear as a dark band (arrow, 15:12) that splits (arrows, 19:00) during anaphase. eGFP-rGBD highlights the centrosomes at all phases of mitosis (arrowhead, 26:00). Times are given in minutes:seconds after filming began. (C and D) Images of rGBD-eGFP (top), TRITC dextran (bottom), and difference images (middle) show that only the signal at the very cortex (C) and at the centrosome (D, arrowheads) is specific to eGFP-rGBD. Images in C are the mean of three successive frames and in D are the mean of 10 successive frames at 3-s intervals. (E and E′) eGFP-rGBD signal intensity measured along the cortex during division in green urchin. E shows raw data from single optical sections 2 min apart (from the cell shown in A), with time points ordered along the rainbow from red to violet. E′ shows curves obtained by fitting a weighted sum of a Gaussian and quadratic to the data in E. The Gaussian fits the furrow signal, whereas the quadratic fits the rest of the cortex. In this and five similar traces we performed, we note that the width of the fit Gaussian varies by 10% or less as the furrow ingresses despite the increasing curvature of the cortex. (F and F′) eGFP-rGBD signal intensity measured along the cortex during division in X. laevis embryo presented as in E and E' except that time points are 20 s apart. (G) Scatter plot of RhoA activity zone width versus cell diameter in urchin embryos; purple squares come from purple urchins; green squares come from green urchins. (H) Scatter plot of RhoA activity zone width versus cell size in X. laevis embryos. (I) Surface view of deconvolved series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in purple urchin embryos; frames are 20 s apart. (J) Z image series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in X. laevis embryos.
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fig2: Characteristics of RhoA activity zones. (A) Single optical sections through a green urchin blastomere before, early, and late in furrowing showing zone in cross section. Bar, 25 μm. (B) Single optical sections through a blastomere from green urchin embryo showing eGFP-rGBD accumulation in spindle region. Chromosomes appear as a dark band (arrow, 15:12) that splits (arrows, 19:00) during anaphase. eGFP-rGBD highlights the centrosomes at all phases of mitosis (arrowhead, 26:00). Times are given in minutes:seconds after filming began. (C and D) Images of rGBD-eGFP (top), TRITC dextran (bottom), and difference images (middle) show that only the signal at the very cortex (C) and at the centrosome (D, arrowheads) is specific to eGFP-rGBD. Images in C are the mean of three successive frames and in D are the mean of 10 successive frames at 3-s intervals. (E and E′) eGFP-rGBD signal intensity measured along the cortex during division in green urchin. E shows raw data from single optical sections 2 min apart (from the cell shown in A), with time points ordered along the rainbow from red to violet. E′ shows curves obtained by fitting a weighted sum of a Gaussian and quadratic to the data in E. The Gaussian fits the furrow signal, whereas the quadratic fits the rest of the cortex. In this and five similar traces we performed, we note that the width of the fit Gaussian varies by 10% or less as the furrow ingresses despite the increasing curvature of the cortex. (F and F′) eGFP-rGBD signal intensity measured along the cortex during division in X. laevis embryo presented as in E and E' except that time points are 20 s apart. (G) Scatter plot of RhoA activity zone width versus cell diameter in urchin embryos; purple squares come from purple urchins; green squares come from green urchins. (H) Scatter plot of RhoA activity zone width versus cell size in X. laevis embryos. (I) Surface view of deconvolved series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in purple urchin embryos; frames are 20 s apart. (J) Z image series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in X. laevis embryos.

Mentions: Further analysis revealed additional features of the cytokinetic RhoA zones. First, the width of the RhoA zone remains nearly constant as furrowing progresses, as shown both by inspection (Fig. 2 A) and by signal intensity analysis of lines drawn along the plasma membrane at successive times during cytokinesis (Fig. 2, E and F). Second, the width of the RhoA zone varied linearly in relation to cell diameter over a range of 5–15 μm (Fig. 2 G) in both species of urchin and frog (Fig. 2 H). Third, a comparison of the timing of eGFP-rGBD accumulation to furrowing onset revealed that the RhoA zone preceded furrowing by 1–4 min (Fig. 2, I and J).


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

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

Characteristics of RhoA activity zones. (A) Single optical sections through a green urchin blastomere before, early, and late in furrowing showing zone in cross section. Bar, 25 μm. (B) Single optical sections through a blastomere from green urchin embryo showing eGFP-rGBD accumulation in spindle region. Chromosomes appear as a dark band (arrow, 15:12) that splits (arrows, 19:00) during anaphase. eGFP-rGBD highlights the centrosomes at all phases of mitosis (arrowhead, 26:00). Times are given in minutes:seconds after filming began. (C and D) Images of rGBD-eGFP (top), TRITC dextran (bottom), and difference images (middle) show that only the signal at the very cortex (C) and at the centrosome (D, arrowheads) is specific to eGFP-rGBD. Images in C are the mean of three successive frames and in D are the mean of 10 successive frames at 3-s intervals. (E and E′) eGFP-rGBD signal intensity measured along the cortex during division in green urchin. E shows raw data from single optical sections 2 min apart (from the cell shown in A), with time points ordered along the rainbow from red to violet. E′ shows curves obtained by fitting a weighted sum of a Gaussian and quadratic to the data in E. The Gaussian fits the furrow signal, whereas the quadratic fits the rest of the cortex. In this and five similar traces we performed, we note that the width of the fit Gaussian varies by 10% or less as the furrow ingresses despite the increasing curvature of the cortex. (F and F′) eGFP-rGBD signal intensity measured along the cortex during division in X. laevis embryo presented as in E and E' except that time points are 20 s apart. (G) Scatter plot of RhoA activity zone width versus cell diameter in urchin embryos; purple squares come from purple urchins; green squares come from green urchins. (H) Scatter plot of RhoA activity zone width versus cell size in X. laevis embryos. (I) Surface view of deconvolved series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in purple urchin embryos; frames are 20 s apart. (J) Z image series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in X. laevis embryos.
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Related In: Results  -  Collection

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fig2: Characteristics of RhoA activity zones. (A) Single optical sections through a green urchin blastomere before, early, and late in furrowing showing zone in cross section. Bar, 25 μm. (B) Single optical sections through a blastomere from green urchin embryo showing eGFP-rGBD accumulation in spindle region. Chromosomes appear as a dark band (arrow, 15:12) that splits (arrows, 19:00) during anaphase. eGFP-rGBD highlights the centrosomes at all phases of mitosis (arrowhead, 26:00). Times are given in minutes:seconds after filming began. (C and D) Images of rGBD-eGFP (top), TRITC dextran (bottom), and difference images (middle) show that only the signal at the very cortex (C) and at the centrosome (D, arrowheads) is specific to eGFP-rGBD. Images in C are the mean of three successive frames and in D are the mean of 10 successive frames at 3-s intervals. (E and E′) eGFP-rGBD signal intensity measured along the cortex during division in green urchin. E shows raw data from single optical sections 2 min apart (from the cell shown in A), with time points ordered along the rainbow from red to violet. E′ shows curves obtained by fitting a weighted sum of a Gaussian and quadratic to the data in E. The Gaussian fits the furrow signal, whereas the quadratic fits the rest of the cortex. In this and five similar traces we performed, we note that the width of the fit Gaussian varies by 10% or less as the furrow ingresses despite the increasing curvature of the cortex. (F and F′) eGFP-rGBD signal intensity measured along the cortex during division in X. laevis embryo presented as in E and E' except that time points are 20 s apart. (G) Scatter plot of RhoA activity zone width versus cell diameter in urchin embryos; purple squares come from purple urchins; green squares come from green urchins. (H) Scatter plot of RhoA activity zone width versus cell size in X. laevis embryos. (I) Surface view of deconvolved series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in purple urchin embryos; frames are 20 s apart. (J) Z image series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in X. laevis embryos.
Mentions: Further analysis revealed additional features of the cytokinetic RhoA zones. First, the width of the RhoA zone remains nearly constant as furrowing progresses, as shown both by inspection (Fig. 2 A) and by signal intensity analysis of lines drawn along the plasma membrane at successive times during cytokinesis (Fig. 2, E and F). Second, the width of the RhoA zone varied linearly in relation to cell diameter over a range of 5–15 μm (Fig. 2 G) in both species of urchin and frog (Fig. 2 H). Third, a comparison of the timing of eGFP-rGBD accumulation to furrowing onset revealed that the RhoA zone preceded furrowing by 1–4 min (Fig. 2, I and J).

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