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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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Midzone-localized signaling complexes become essential for furrow formation when aster separation is delayed. (A) Confocal images of tpxl-1(RNAi), zen-4 & tpxl-1(RNAi), and air-2 & tpxl-1(RNAi) embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe. (B) The mean aster-to-aster distance, measured from the sequences in A, is plotted versus time in seconds after NEBD for each condition. The control and tpxl-1(RNAi) curves from Figure 1B are reproduced here for comparison. Error bars are SEM. (C) Central plane confocal images of embryos expressing a GFP plasma membrane probe. Montages of the equatorial region are shown: furrow involution is indicated (purple arrowheads). The number of embryos forming a double membrane furrow is indicated. Times are in seconds after NEBD. Bars, 10 μm.
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Figure 7: Midzone-localized signaling complexes become essential for furrow formation when aster separation is delayed. (A) Confocal images of tpxl-1(RNAi), zen-4 & tpxl-1(RNAi), and air-2 & tpxl-1(RNAi) embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe. (B) The mean aster-to-aster distance, measured from the sequences in A, is plotted versus time in seconds after NEBD for each condition. The control and tpxl-1(RNAi) curves from Figure 1B are reproduced here for comparison. Error bars are SEM. (C) Central plane confocal images of embryos expressing a GFP plasma membrane probe. Montages of the equatorial region are shown: furrow involution is indicated (purple arrowheads). The number of embryos forming a double membrane furrow is indicated. Times are in seconds after NEBD. Bars, 10 μm.

Mentions: The opposite phenotypes resulting from delaying aster separation and inhibiting midzone signaling suggest that simultaneous inhibition of aster separation and midzone signaling would abolish furrow formation. To test this idea, we codepleted TPXL-1 and either ZEN-4 or Aurora BAIR-2. The kinetics of aster separation in the codepleted embryos were similar to those in embryos depleted of TPXL-1 alone (Figure 7, A and B); however, furrow involution was abolished (Figure 7C and Supplemental Movie S5). This suggests that when aster separation is delayed, furrow formation becomes dependent on centralspindlin and the CPC.


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)

Midzone-localized signaling complexes become essential for furrow formation when aster separation is delayed. (A) Confocal images of tpxl-1(RNAi), zen-4 & tpxl-1(RNAi), and air-2 & tpxl-1(RNAi) embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe. (B) The mean aster-to-aster distance, measured from the sequences in A, is plotted versus time in seconds after NEBD for each condition. The control and tpxl-1(RNAi) curves from Figure 1B are reproduced here for comparison. Error bars are SEM. (C) Central plane confocal images of embryos expressing a GFP plasma membrane probe. Montages of the equatorial region are shown: furrow involution is indicated (purple arrowheads). The number of embryos forming a double membrane furrow is indicated. Times are in seconds after NEBD. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 7: Midzone-localized signaling complexes become essential for furrow formation when aster separation is delayed. (A) Confocal images of tpxl-1(RNAi), zen-4 & tpxl-1(RNAi), and air-2 & tpxl-1(RNAi) embryos coexpressing GFP:β-tubulin and a GFP plasma membrane probe. (B) The mean aster-to-aster distance, measured from the sequences in A, is plotted versus time in seconds after NEBD for each condition. The control and tpxl-1(RNAi) curves from Figure 1B are reproduced here for comparison. Error bars are SEM. (C) Central plane confocal images of embryos expressing a GFP plasma membrane probe. Montages of the equatorial region are shown: furrow involution is indicated (purple arrowheads). The number of embryos forming a double membrane furrow is indicated. Times are in seconds after NEBD. Bars, 10 μm.
Mentions: The opposite phenotypes resulting from delaying aster separation and inhibiting midzone signaling suggest that simultaneous inhibition of aster separation and midzone signaling would abolish furrow formation. To test this idea, we codepleted TPXL-1 and either ZEN-4 or Aurora BAIR-2. The kinetics of aster separation in the codepleted embryos were similar to those in embryos depleted of TPXL-1 alone (Figure 7, A and B); however, furrow involution was abolished (Figure 7C and Supplemental Movie S5). This suggests that when aster separation is delayed, furrow formation becomes dependent on centralspindlin and the CPC.

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