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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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Aster separation is required for the equatorial enrichment of contractile ring proteins following anaphase onset. (A) Images of the cortex in embryos expressing GFP:Anillin. Images are maximum intensity projections of four z-sections collected at 1-μm intervals (see schematic). (B) Schematic illustrating the method used to analyze cortical GFP:Anillin distribution. A 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal length segments from anterior (0%) to posterior (100%). (C) The mean postanaphase accumulation of cortical GFP:Anillin is plotted as a function of embryo length. The mean GFP:Anillin in each segment, after subtraction of a background measurement made before anaphase onset, is plotted for each time point. Values were normalized by dividing by the average maximum value for controls. In TPXL-1–depleted embryos, a slight enrichment of cortical GFP:Anillin is observed on the polar cortices (arrowheads in graphs for 240- and 280-s time points) relative to the equator. Error bars are SEM (D) Examples of cortical GFP:Anillin accumulation in control and tpxl-1(RNAi) embryos 240 s after NEBD. Boxed regions are magnified 2×. Times are in seconds after NEBD. Bars, 10 μm.
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Figure 3: Aster separation is required for the equatorial enrichment of contractile ring proteins following anaphase onset. (A) Images of the cortex in embryos expressing GFP:Anillin. Images are maximum intensity projections of four z-sections collected at 1-μm intervals (see schematic). (B) Schematic illustrating the method used to analyze cortical GFP:Anillin distribution. A 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal length segments from anterior (0%) to posterior (100%). (C) The mean postanaphase accumulation of cortical GFP:Anillin is plotted as a function of embryo length. The mean GFP:Anillin in each segment, after subtraction of a background measurement made before anaphase onset, is plotted for each time point. Values were normalized by dividing by the average maximum value for controls. In TPXL-1–depleted embryos, a slight enrichment of cortical GFP:Anillin is observed on the polar cortices (arrowheads in graphs for 240- and 280-s time points) relative to the equator. Error bars are SEM (D) Examples of cortical GFP:Anillin accumulation in control and tpxl-1(RNAi) embryos 240 s after NEBD. Boxed regions are magnified 2×. Times are in seconds after NEBD. Bars, 10 μm.

Mentions: To understand how delaying aster separation delays furrow formation, we next examined the consequences of delaying aster separation on the cortical accumulation of GFP fusions with two contractile ring components, Anillin (GFP:AnillinANI-1; Figure 3 and Supplemental Movie S3) and the heavy chain of myosin II (NMY-2:GFP; Supplemental Figure S2). The postanaphase pattern of equatorial accumulation was essentially identical for the two markers (Figure 3, A–C, and Supplemental Figure S2, A and B). In control embryos, GFP:Anillin and NMY-2:GFP accumulated on the equatorial cortex during the 100 s after anaphase onset (180–280 s after NEBD), forming a band that peaked at ∼60% of embryo length. Furrow involution occurred ∼10 s after the equatorial band reached its maximal intensity (Figure 3C; 280 s). In TPXL-1–depleted embryos, GFP:Anillin and NMY-2:GFP also accumulated on the cortex during the 100 s after anaphase onset. However, instead of being concentrated at the cell equator, patches of GFP:Anillin and NMY-2:GFP were distributed over the entire cortex (Figure 3, A and D; Supplemental Figure S2, A and C; and Supplemental Movie S3). Quantitative analysis revealed a slight enrichment of cortical GFP:Anillin on the anterior and posterior cortices relative to the cell equator (Figure 3C, arrowheads in graphs for 240- and 280s time points)—a pattern inverse to that seen in controls at this time. Thus, separated asters are critical for the equatorial enrichment of contractile ring proteins after anaphase onset.


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)

Aster separation is required for the equatorial enrichment of contractile ring proteins following anaphase onset. (A) Images of the cortex in embryos expressing GFP:Anillin. Images are maximum intensity projections of four z-sections collected at 1-μm intervals (see schematic). (B) Schematic illustrating the method used to analyze cortical GFP:Anillin distribution. A 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal length segments from anterior (0%) to posterior (100%). (C) The mean postanaphase accumulation of cortical GFP:Anillin is plotted as a function of embryo length. The mean GFP:Anillin in each segment, after subtraction of a background measurement made before anaphase onset, is plotted for each time point. Values were normalized by dividing by the average maximum value for controls. In TPXL-1–depleted embryos, a slight enrichment of cortical GFP:Anillin is observed on the polar cortices (arrowheads in graphs for 240- and 280-s time points) relative to the equator. Error bars are SEM (D) Examples of cortical GFP:Anillin accumulation in control and tpxl-1(RNAi) embryos 240 s after NEBD. Boxed regions are magnified 2×. Times are in seconds after NEBD. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Aster separation is required for the equatorial enrichment of contractile ring proteins following anaphase onset. (A) Images of the cortex in embryos expressing GFP:Anillin. Images are maximum intensity projections of four z-sections collected at 1-μm intervals (see schematic). (B) Schematic illustrating the method used to analyze cortical GFP:Anillin distribution. A 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal length segments from anterior (0%) to posterior (100%). (C) The mean postanaphase accumulation of cortical GFP:Anillin is plotted as a function of embryo length. The mean GFP:Anillin in each segment, after subtraction of a background measurement made before anaphase onset, is plotted for each time point. Values were normalized by dividing by the average maximum value for controls. In TPXL-1–depleted embryos, a slight enrichment of cortical GFP:Anillin is observed on the polar cortices (arrowheads in graphs for 240- and 280-s time points) relative to the equator. Error bars are SEM (D) Examples of cortical GFP:Anillin accumulation in control and tpxl-1(RNAi) embryos 240 s after NEBD. Boxed regions are magnified 2×. Times are in seconds after NEBD. Bars, 10 μm.
Mentions: To understand how delaying aster separation delays furrow formation, we next examined the consequences of delaying aster separation on the cortical accumulation of GFP fusions with two contractile ring components, Anillin (GFP:AnillinANI-1; Figure 3 and Supplemental Movie S3) and the heavy chain of myosin II (NMY-2:GFP; Supplemental Figure S2). The postanaphase pattern of equatorial accumulation was essentially identical for the two markers (Figure 3, A–C, and Supplemental Figure S2, A and B). In control embryos, GFP:Anillin and NMY-2:GFP accumulated on the equatorial cortex during the 100 s after anaphase onset (180–280 s after NEBD), forming a band that peaked at ∼60% of embryo length. Furrow involution occurred ∼10 s after the equatorial band reached its maximal intensity (Figure 3C; 280 s). In TPXL-1–depleted embryos, GFP:Anillin and NMY-2:GFP also accumulated on the cortex during the 100 s after anaphase onset. However, instead of being concentrated at the cell equator, patches of GFP:Anillin and NMY-2:GFP were distributed over the entire cortex (Figure 3, A and D; Supplemental Figure S2, A and C; and Supplemental Movie S3). Quantitative analysis revealed a slight enrichment of cortical GFP:Anillin on the anterior and posterior cortices relative to the cell equator (Figure 3C, arrowheads in graphs for 240- and 280s time points)—a pattern inverse to that seen in controls at this time. Thus, separated asters are critical for the equatorial enrichment of contractile ring proteins after anaphase onset.

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