Limits...
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.

Show MeSH

Related in: MedlinePlus

GFP:Anillin accumulates on the equatorial cortex with normal timing after depletion of SPD-1, ZEN-4, or AuroraBAIR-2. (A) Spinning disk confocal optics were used to image the cortex in control (n = 46), spd-1(RNAi) (n = 10), zen-4(RNAi) (n = 13), and air-2(RNAi) (n = 10) embryos expressing GFP:Anillin. Images are maximum intensity projections of four cortical sections collected at 1-μm z intervals. (B) The mean postanaphase accumulation of cortical GFP:Anillin was quantified as a function of embryo length (as described for Figure 3) for control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos at the indicated time points after NEBD. Error bars are SEM. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2801719&req=5

Figure 6: GFP:Anillin accumulates on the equatorial cortex with normal timing after depletion of SPD-1, ZEN-4, or AuroraBAIR-2. (A) Spinning disk confocal optics were used to image the cortex in control (n = 46), spd-1(RNAi) (n = 10), zen-4(RNAi) (n = 13), and air-2(RNAi) (n = 10) embryos expressing GFP:Anillin. Images are maximum intensity projections of four cortical sections collected at 1-μm z intervals. (B) The mean postanaphase accumulation of cortical GFP:Anillin was quantified as a function of embryo length (as described for Figure 3) for control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos at the indicated time points after NEBD. Error bars are SEM. Bar, 10 μm.

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)

GFP:Anillin accumulates on the equatorial cortex with normal timing after depletion of SPD-1, ZEN-4, or AuroraBAIR-2. (A) Spinning disk confocal optics were used to image the cortex in control (n = 46), spd-1(RNAi) (n = 10), zen-4(RNAi) (n = 13), and air-2(RNAi) (n = 10) embryos expressing GFP:Anillin. Images are maximum intensity projections of four cortical sections collected at 1-μm z intervals. (B) The mean postanaphase accumulation of cortical GFP:Anillin was quantified as a function of embryo length (as described for Figure 3) for control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos at the indicated time points after NEBD. Error bars are SEM. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801719&req=5

Figure 6: GFP:Anillin accumulates on the equatorial cortex with normal timing after depletion of SPD-1, ZEN-4, or AuroraBAIR-2. (A) Spinning disk confocal optics were used to image the cortex in control (n = 46), spd-1(RNAi) (n = 10), zen-4(RNAi) (n = 13), and air-2(RNAi) (n = 10) embryos expressing GFP:Anillin. Images are maximum intensity projections of four cortical sections collected at 1-μm z intervals. (B) The mean postanaphase accumulation of cortical GFP:Anillin was quantified as a function of embryo length (as described for Figure 3) for control, spd-1(RNAi), zen-4(RNAi), and air-2(RNAi) embryos at the indicated time points after NEBD. Error bars are SEM. Bar, 10 μm.
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