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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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TPXL-1 depletion does not significantly alter the localization of midzone-localized signaling complexes. (A) Fixed control and tpxl-1(RNAi) embryos were stained to visualize DNA, α-tubulin, ZEN-4, and AIR-2. 3D widefield data stacks were collected and computationally deconvolved. Maximum intensity projections of the central region of representative early (top) and late (bottom) anaphase embryos are shown. (B) Single-plane confocal images of the spindle in embryos expressing GFP:AuroraBAIR-2 and mCherry:Histone H2B. Times are relative to anaphase onset. Graph shows the average GFP:AuroraBAIR-2 fluorescence intensity as a function of position along the spindle axis (see diagram) in control and tpxl-1(RNAi) embryos 120 s after anaphase onset, which corresponds to the time of furrow involution in controls. Values are plotted over the length of the midzone, with the zero position corresponding to the fluorescence intensity maximum. Error bars are the SD. Bars, 10 μm.
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Figure 4: TPXL-1 depletion does not significantly alter the localization of midzone-localized signaling complexes. (A) Fixed control and tpxl-1(RNAi) embryos were stained to visualize DNA, α-tubulin, ZEN-4, and AIR-2. 3D widefield data stacks were collected and computationally deconvolved. Maximum intensity projections of the central region of representative early (top) and late (bottom) anaphase embryos are shown. (B) Single-plane confocal images of the spindle in embryos expressing GFP:AuroraBAIR-2 and mCherry:Histone H2B. Times are relative to anaphase onset. Graph shows the average GFP:AuroraBAIR-2 fluorescence intensity as a function of position along the spindle axis (see diagram) in control and tpxl-1(RNAi) embryos 120 s after anaphase onset, which corresponds to the time of furrow involution in controls. Values are plotted over the length of the midzone, with the zero position corresponding to the fluorescence intensity maximum. Error bars are the SD. Bars, 10 μm.

Mentions: The graph in Figure 4B was generated by drawing a 40-pixel wide box along the spindle axis in images of control and tpxl-1(RNAi) embryos acquired 120 s after anaphase onset and generating a linescan of average fluorescence intensity versus position along the spindle axis. After subtracting the cytoplasmic background (average of the final 5 values of the linescan, nearest the embryo posterior) from all values, the traces from five to six individual embryos were aligned by setting the fluorescence intensity maximum for each scan to position = 0. After aligning, the average fluorescence intensity at each position along the spindle axis was calculated for the set of scans and plotted.


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)

TPXL-1 depletion does not significantly alter the localization of midzone-localized signaling complexes. (A) Fixed control and tpxl-1(RNAi) embryos were stained to visualize DNA, α-tubulin, ZEN-4, and AIR-2. 3D widefield data stacks were collected and computationally deconvolved. Maximum intensity projections of the central region of representative early (top) and late (bottom) anaphase embryos are shown. (B) Single-plane confocal images of the spindle in embryos expressing GFP:AuroraBAIR-2 and mCherry:Histone H2B. Times are relative to anaphase onset. Graph shows the average GFP:AuroraBAIR-2 fluorescence intensity as a function of position along the spindle axis (see diagram) in control and tpxl-1(RNAi) embryos 120 s after anaphase onset, which corresponds to the time of furrow involution in controls. Values are plotted over the length of the midzone, with the zero position corresponding to the fluorescence intensity maximum. Error bars are the SD. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: TPXL-1 depletion does not significantly alter the localization of midzone-localized signaling complexes. (A) Fixed control and tpxl-1(RNAi) embryos were stained to visualize DNA, α-tubulin, ZEN-4, and AIR-2. 3D widefield data stacks were collected and computationally deconvolved. Maximum intensity projections of the central region of representative early (top) and late (bottom) anaphase embryos are shown. (B) Single-plane confocal images of the spindle in embryos expressing GFP:AuroraBAIR-2 and mCherry:Histone H2B. Times are relative to anaphase onset. Graph shows the average GFP:AuroraBAIR-2 fluorescence intensity as a function of position along the spindle axis (see diagram) in control and tpxl-1(RNAi) embryos 120 s after anaphase onset, which corresponds to the time of furrow involution in controls. Values are plotted over the length of the midzone, with the zero position corresponding to the fluorescence intensity maximum. Error bars are the SD. Bars, 10 μm.
Mentions: The graph in Figure 4B was generated by drawing a 40-pixel wide box along the spindle axis in images of control and tpxl-1(RNAi) embryos acquired 120 s after anaphase onset and generating a linescan of average fluorescence intensity versus position along the spindle axis. After subtracting the cytoplasmic background (average of the final 5 values of the linescan, nearest the embryo posterior) from all values, the traces from five to six individual embryos were aligned by setting the fluorescence intensity maximum for each scan to position = 0. After aligning, the average fluorescence intensity at each position along the spindle axis was calculated for the set of scans and plotted.

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