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Analyzing the effects of delaying aster separation on furrow formation during cytokinesis in the Caenorhabditis elegans embryo.

Lewellyn L, Dumont J, Desai A, Oegema K - Mol. Biol. Cell (2009)

Bottom Line: Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow.Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold.Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow. Here, we explore the contribution of the asters by analyzing the consequences of altering interaster distance during the first cytokinesis of the Caenorhabditis elegans embryo. Delaying aster separation, by using TPXL-1 depletion to shorten the metaphase spindle, leads to a corresponding delay in furrow formation, but results in a single furrow that ingresses at a normal rate. Preventing aster separation, by simultaneously inhibiting TPXL-1 and Galpha signaling-based cortical forces pulling on the asters, delays furrow formation and leads to the formation of multiple furrows that ingress toward the midzone. Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold. Simultaneously delaying aster separation and disrupting midzone-based signaling leads to complete failure of furrow formation. Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.

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Furrows form with normal timing but ingress at a reduced rate after inhibition of centralspindlin or the chromosomal passenger complex. (A) Schematics summarize the effects of each perturbation on chromosome segregation and anaphase spindle structure. The mean aster-to-aster distance, measured from confocal images of embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted versus time in seconds after NEBD for each condition. The control curve from Figure 1B is reproduced for comparison. Dotted line indicates anaphase onset in controls. Error bars are SEM. (B) The interval between NEBD and furrow involution, measured in embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted for individual embryos. The data for the control embryos is reproduced from Figure 2B for comparison. Solid line indicates the mean. Dashed line indicates the mean time of anaphase onset in controls. (C) Spinning disk confocal optics were used to acquire z-series of control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos expressing the GFP plasma membrane probe, and reconstructions yielding an “end-on” view of the division plane were generated. Representative projections from time-lapse series are shown for each condition. The fraction of embryos that completed the first cytokinesis is indicated to the right. Bar, 10 μm. (D) Mean furrow diameter was measured from end on reconstructions generated as in C and is plotted versus time. The control curve is reproduced from Figure 2D for comparison. Times in C and D are in seconds after furrow initiation, the time when the first detectable ingression was visible. Error bars are the 90% confidence interval for the mean. Double asterisks indicate furrow regression.
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Figure 5: Furrows form with normal timing but ingress at a reduced rate after inhibition of centralspindlin or the chromosomal passenger complex. (A) Schematics summarize the effects of each perturbation on chromosome segregation and anaphase spindle structure. The mean aster-to-aster distance, measured from confocal images of embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted versus time in seconds after NEBD for each condition. The control curve from Figure 1B is reproduced for comparison. Dotted line indicates anaphase onset in controls. Error bars are SEM. (B) The interval between NEBD and furrow involution, measured in embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted for individual embryos. The data for the control embryos is reproduced from Figure 2B for comparison. Solid line indicates the mean. Dashed line indicates the mean time of anaphase onset in controls. (C) Spinning disk confocal optics were used to acquire z-series of control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos expressing the GFP plasma membrane probe, and reconstructions yielding an “end-on” view of the division plane were generated. Representative projections from time-lapse series are shown for each condition. The fraction of embryos that completed the first cytokinesis is indicated to the right. Bar, 10 μm. (D) Mean furrow diameter was measured from end on reconstructions generated as in C and is plotted versus time. The control curve is reproduced from Figure 2D for comparison. Times in C and D are in seconds after furrow initiation, the time when the first detectable ingression was visible. Error bars are the 90% confidence interval for the mean. Double asterisks indicate furrow regression.

Mentions: To understand the relative contributions of signaling by the asters and midzone during cytokinesis, we tested whether inhibition of midzone-localized proteins would result in a delay in furrow formation comparable with that resulting from delaying aster separation. Using the methods we developed to characterize TPXL-1–depleted embryos, we analyzed the phenotype of embryos depleted of three different midzone proteins—the centralspindlin component ZEN-4, the CPC component AuroraBAIR-2, and SPD-1, a microtubule-binding protein required for the stable bundling of midzone microtubules (Vebrugghe and White, 2004). Depletion of any of these midzone components disrupts the formation of midzone microtubule bundles and leads to premature spindle pole separation (Figure 5A). Depletion of AuroraBAIR-2 additionally results in defects in meiotic and mitotic chromosome segregation, which probably underlies the different aster separation kinetics for this depletion compared with depletion of SPD-1 or ZEN-4 (Figure 5A). Although midzone microtubules failed to form stable bundles (Supplemental Figure S3), the equatorial recruitment of contractile ring proteins and furrow formation occurred with normal timing in SPD-1–depleted embryos (Figures 5B and 6, A and B), and the rate of furrow ingression and success of the first cytokinesis were not different from controls (Figure 5, C and D). The ability of SPD-1–depleted embryos to undergo a normal division may be due to the fact that the CPC and centralspindlin continue to localize to microtubules in the midzone region, despite the failure to form stable microtubule bundles (Supplemental Figure S3).


Analyzing the effects of delaying aster separation on furrow formation during cytokinesis in the Caenorhabditis elegans embryo.

Lewellyn L, Dumont J, Desai A, Oegema K - Mol. Biol. Cell (2009)

Furrows form with normal timing but ingress at a reduced rate after inhibition of centralspindlin or the chromosomal passenger complex. (A) Schematics summarize the effects of each perturbation on chromosome segregation and anaphase spindle structure. The mean aster-to-aster distance, measured from confocal images of embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted versus time in seconds after NEBD for each condition. The control curve from Figure 1B is reproduced for comparison. Dotted line indicates anaphase onset in controls. Error bars are SEM. (B) The interval between NEBD and furrow involution, measured in embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted for individual embryos. The data for the control embryos is reproduced from Figure 2B for comparison. Solid line indicates the mean. Dashed line indicates the mean time of anaphase onset in controls. (C) Spinning disk confocal optics were used to acquire z-series of control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos expressing the GFP plasma membrane probe, and reconstructions yielding an “end-on” view of the division plane were generated. Representative projections from time-lapse series are shown for each condition. The fraction of embryos that completed the first cytokinesis is indicated to the right. Bar, 10 μm. (D) Mean furrow diameter was measured from end on reconstructions generated as in C and is plotted versus time. The control curve is reproduced from Figure 2D for comparison. Times in C and D are in seconds after furrow initiation, the time when the first detectable ingression was visible. Error bars are the 90% confidence interval for the mean. Double asterisks indicate furrow regression.
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Related In: Results  -  Collection

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Figure 5: Furrows form with normal timing but ingress at a reduced rate after inhibition of centralspindlin or the chromosomal passenger complex. (A) Schematics summarize the effects of each perturbation on chromosome segregation and anaphase spindle structure. The mean aster-to-aster distance, measured from confocal images of embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted versus time in seconds after NEBD for each condition. The control curve from Figure 1B is reproduced for comparison. Dotted line indicates anaphase onset in controls. Error bars are SEM. (B) The interval between NEBD and furrow involution, measured in embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe, is plotted for individual embryos. The data for the control embryos is reproduced from Figure 2B for comparison. Solid line indicates the mean. Dashed line indicates the mean time of anaphase onset in controls. (C) Spinning disk confocal optics were used to acquire z-series of control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos expressing the GFP plasma membrane probe, and reconstructions yielding an “end-on” view of the division plane were generated. Representative projections from time-lapse series are shown for each condition. The fraction of embryos that completed the first cytokinesis is indicated to the right. Bar, 10 μm. (D) Mean furrow diameter was measured from end on reconstructions generated as in C and is plotted versus time. The control curve is reproduced from Figure 2D for comparison. Times in C and D are in seconds after furrow initiation, the time when the first detectable ingression was visible. Error bars are the 90% confidence interval for the mean. Double asterisks indicate furrow regression.
Mentions: To understand the relative contributions of signaling by the asters and midzone during cytokinesis, we tested whether inhibition of midzone-localized proteins would result in a delay in furrow formation comparable with that resulting from delaying aster separation. Using the methods we developed to characterize TPXL-1–depleted embryos, we analyzed the phenotype of embryos depleted of three different midzone proteins—the centralspindlin component ZEN-4, the CPC component AuroraBAIR-2, and SPD-1, a microtubule-binding protein required for the stable bundling of midzone microtubules (Vebrugghe and White, 2004). Depletion of any of these midzone components disrupts the formation of midzone microtubule bundles and leads to premature spindle pole separation (Figure 5A). Depletion of AuroraBAIR-2 additionally results in defects in meiotic and mitotic chromosome segregation, which probably underlies the different aster separation kinetics for this depletion compared with depletion of SPD-1 or ZEN-4 (Figure 5A). Although midzone microtubules failed to form stable bundles (Supplemental Figure S3), the equatorial recruitment of contractile ring proteins and furrow formation occurred with normal timing in SPD-1–depleted embryos (Figures 5B and 6, A and B), and the rate of furrow ingression and success of the first cytokinesis were not different from controls (Figure 5, C and D). The ability of SPD-1–depleted embryos to undergo a normal division may be due to the fact that the CPC and centralspindlin continue to localize to microtubules in the midzone region, despite the failure to form stable microtubule bundles (Supplemental Figure S3).

Bottom Line: Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow.Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold.Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow. Here, we explore the contribution of the asters by analyzing the consequences of altering interaster distance during the first cytokinesis of the Caenorhabditis elegans embryo. Delaying aster separation, by using TPXL-1 depletion to shorten the metaphase spindle, leads to a corresponding delay in furrow formation, but results in a single furrow that ingresses at a normal rate. Preventing aster separation, by simultaneously inhibiting TPXL-1 and Galpha signaling-based cortical forces pulling on the asters, delays furrow formation and leads to the formation of multiple furrows that ingress toward the midzone. Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold. Simultaneously delaying aster separation and disrupting midzone-based signaling leads to complete failure of furrow formation. Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.

Show MeSH
Related in: MedlinePlus