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Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle.

Lee KY, Esmaeili B, Zealley B, Mishima M - Nat Commun (2015)

Bottom Line: The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis.How the cell balances these potentially conflicting requirements is poorly understood.This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

View Article: PubMed Central - PubMed

Affiliation: 1] Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK [2] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

ABSTRACT
During animal cell division, the central spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the central spindle is the only structure linking the two halves of the mitotic spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine spindle positioning and drive chromosome segregation through spindle elongation. The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the central spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

No MeSH data available.


Related in: MedlinePlus

Resilience of central spindle against excess tension.The central spindle in C. elegans can recover from near disruption during the first embryonic division. (a) Schematic of an animal cell in anaphase. The central spindle is under mechanical tension from cortical pulling forces. (b) Spinning disk confocal time-lapse images of embryos expressing mCherry::tubulin (magenta) and a major organizer of the central spindle, CYK-4::GFP (green), after the depletion of EFA-6, BMK-1 and SPD-1 by RNAi. The arrowheads indicate the sudden broadening of CYK-4 midzone accumulation associated with the mid-anaphase acceleration of pole-to-pole elongation caused by the depletion of these molecules. The central spindle was not completely broken in efa-6(RNAi) (enhanced outward pulling force) or in bmk-1(RNAi) (less drag against the pulling force) embryos, and CYK-4 accumulation was later restored. This recovery did not occur in embryos depleted of SPD-1/PRC1, another major central spindle organizer. The midzone accumulation of CYK-4 was largely lost (arrow), although weak accumulation via the furrow-dependent pathway35 was observed later (double-headed arrow). Scale bar, 10 μm. (c, d) Quantitative analysis of morphological changes in the mitotic spindle. The positions of the spindle poles were determined as the peak mCherry::tubulin signals, and the distance between them (pole-to-pole distance) is plotted in magenta. The intensity of CYK-4::GFP in individual embryos was standardized using a uniform cytoplasmic signal during the pre-mitotic stage. Line profiles of 4.4 μm in width between the two poles were measured for each time point, and the width at half-maximum (CYK-4 peak width), height (CYK-4 peak intensity) and area (total CYK-4 on spindle) of the peak were measured and are plotted in green. The dotted lines on the graphs of CYK-4 peak width indicate the pole-to-pole distance during metaphase. In the efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi) panels, the values for the control are also plotted in grey. Recovery after near disruption in efa-6(RNAi) and bmk-1(RNAi) embryos was detected as gradual tightening of the CYK-4 peak width (arrows) after sudden widening (arrowheads). The grey arrows indicate the loss of CYK-4 accumulation in spd-1(RNAi) embryos and the central spindle-independent late recruitment. The mean and standard error were determined from 33, 7, 6 and 6 embryos for the control, efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi), respectively.
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f1: Resilience of central spindle against excess tension.The central spindle in C. elegans can recover from near disruption during the first embryonic division. (a) Schematic of an animal cell in anaphase. The central spindle is under mechanical tension from cortical pulling forces. (b) Spinning disk confocal time-lapse images of embryos expressing mCherry::tubulin (magenta) and a major organizer of the central spindle, CYK-4::GFP (green), after the depletion of EFA-6, BMK-1 and SPD-1 by RNAi. The arrowheads indicate the sudden broadening of CYK-4 midzone accumulation associated with the mid-anaphase acceleration of pole-to-pole elongation caused by the depletion of these molecules. The central spindle was not completely broken in efa-6(RNAi) (enhanced outward pulling force) or in bmk-1(RNAi) (less drag against the pulling force) embryos, and CYK-4 accumulation was later restored. This recovery did not occur in embryos depleted of SPD-1/PRC1, another major central spindle organizer. The midzone accumulation of CYK-4 was largely lost (arrow), although weak accumulation via the furrow-dependent pathway35 was observed later (double-headed arrow). Scale bar, 10 μm. (c, d) Quantitative analysis of morphological changes in the mitotic spindle. The positions of the spindle poles were determined as the peak mCherry::tubulin signals, and the distance between them (pole-to-pole distance) is plotted in magenta. The intensity of CYK-4::GFP in individual embryos was standardized using a uniform cytoplasmic signal during the pre-mitotic stage. Line profiles of 4.4 μm in width between the two poles were measured for each time point, and the width at half-maximum (CYK-4 peak width), height (CYK-4 peak intensity) and area (total CYK-4 on spindle) of the peak were measured and are plotted in green. The dotted lines on the graphs of CYK-4 peak width indicate the pole-to-pole distance during metaphase. In the efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi) panels, the values for the control are also plotted in grey. Recovery after near disruption in efa-6(RNAi) and bmk-1(RNAi) embryos was detected as gradual tightening of the CYK-4 peak width (arrows) after sudden widening (arrowheads). The grey arrows indicate the loss of CYK-4 accumulation in spd-1(RNAi) embryos and the central spindle-independent late recruitment. The mean and standard error were determined from 33, 7, 6 and 6 embryos for the control, efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi), respectively.

Mentions: To examine the response of the central spindle to increased pulling forces (Fig. 1a), we generated transgenic C. elegans strains expressing mCherry-tagged tubulin and green fluorescent protein (GFP)-tagged CYK-4. During the first mitotic division of control embryos, the bipolar spindle formed promptly following nuclear envelope breakdown (NEBD, time=0). NEBD was monitored by the loss of exclusion of the tubulin signal from pronuclei (Fig. 1b and Supplementary Fig. 1 for grayscale images covering the whole embryos). Approximately 120–150 s following NEBD, the distance between the two spindle poles (Fig. 1c) started to increase, indicating the onset of anaphase, as previously reported30 (Fig. 1d). At the same time, CYK-4 began to accumulate at the spindle midzone (Fig. 1b, control), where the plus ends of the two sets of interpolar microtubules from the two spindle poles form an interdigitating overlap, as confirmed by an accelerated increase in the peak intensity (Fig. 1d, control and Supplementary Fig. 2a for examples of the line profiling of CYK-4::GFP intensity). Within the next 30–60 s, the central spindle was established, with CYK-4 enriching rapidly in the central overlap zone and remaining there for ∼300 s until the cleavage furrow fully ingressed and the central spindle was compacted to form the midbody.


Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle.

Lee KY, Esmaeili B, Zealley B, Mishima M - Nat Commun (2015)

Resilience of central spindle against excess tension.The central spindle in C. elegans can recover from near disruption during the first embryonic division. (a) Schematic of an animal cell in anaphase. The central spindle is under mechanical tension from cortical pulling forces. (b) Spinning disk confocal time-lapse images of embryos expressing mCherry::tubulin (magenta) and a major organizer of the central spindle, CYK-4::GFP (green), after the depletion of EFA-6, BMK-1 and SPD-1 by RNAi. The arrowheads indicate the sudden broadening of CYK-4 midzone accumulation associated with the mid-anaphase acceleration of pole-to-pole elongation caused by the depletion of these molecules. The central spindle was not completely broken in efa-6(RNAi) (enhanced outward pulling force) or in bmk-1(RNAi) (less drag against the pulling force) embryos, and CYK-4 accumulation was later restored. This recovery did not occur in embryos depleted of SPD-1/PRC1, another major central spindle organizer. The midzone accumulation of CYK-4 was largely lost (arrow), although weak accumulation via the furrow-dependent pathway35 was observed later (double-headed arrow). Scale bar, 10 μm. (c, d) Quantitative analysis of morphological changes in the mitotic spindle. The positions of the spindle poles were determined as the peak mCherry::tubulin signals, and the distance between them (pole-to-pole distance) is plotted in magenta. The intensity of CYK-4::GFP in individual embryos was standardized using a uniform cytoplasmic signal during the pre-mitotic stage. Line profiles of 4.4 μm in width between the two poles were measured for each time point, and the width at half-maximum (CYK-4 peak width), height (CYK-4 peak intensity) and area (total CYK-4 on spindle) of the peak were measured and are plotted in green. The dotted lines on the graphs of CYK-4 peak width indicate the pole-to-pole distance during metaphase. In the efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi) panels, the values for the control are also plotted in grey. Recovery after near disruption in efa-6(RNAi) and bmk-1(RNAi) embryos was detected as gradual tightening of the CYK-4 peak width (arrows) after sudden widening (arrowheads). The grey arrows indicate the loss of CYK-4 accumulation in spd-1(RNAi) embryos and the central spindle-independent late recruitment. The mean and standard error were determined from 33, 7, 6 and 6 embryos for the control, efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi), respectively.
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Related In: Results  -  Collection

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f1: Resilience of central spindle against excess tension.The central spindle in C. elegans can recover from near disruption during the first embryonic division. (a) Schematic of an animal cell in anaphase. The central spindle is under mechanical tension from cortical pulling forces. (b) Spinning disk confocal time-lapse images of embryos expressing mCherry::tubulin (magenta) and a major organizer of the central spindle, CYK-4::GFP (green), after the depletion of EFA-6, BMK-1 and SPD-1 by RNAi. The arrowheads indicate the sudden broadening of CYK-4 midzone accumulation associated with the mid-anaphase acceleration of pole-to-pole elongation caused by the depletion of these molecules. The central spindle was not completely broken in efa-6(RNAi) (enhanced outward pulling force) or in bmk-1(RNAi) (less drag against the pulling force) embryos, and CYK-4 accumulation was later restored. This recovery did not occur in embryos depleted of SPD-1/PRC1, another major central spindle organizer. The midzone accumulation of CYK-4 was largely lost (arrow), although weak accumulation via the furrow-dependent pathway35 was observed later (double-headed arrow). Scale bar, 10 μm. (c, d) Quantitative analysis of morphological changes in the mitotic spindle. The positions of the spindle poles were determined as the peak mCherry::tubulin signals, and the distance between them (pole-to-pole distance) is plotted in magenta. The intensity of CYK-4::GFP in individual embryos was standardized using a uniform cytoplasmic signal during the pre-mitotic stage. Line profiles of 4.4 μm in width between the two poles were measured for each time point, and the width at half-maximum (CYK-4 peak width), height (CYK-4 peak intensity) and area (total CYK-4 on spindle) of the peak were measured and are plotted in green. The dotted lines on the graphs of CYK-4 peak width indicate the pole-to-pole distance during metaphase. In the efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi) panels, the values for the control are also plotted in grey. Recovery after near disruption in efa-6(RNAi) and bmk-1(RNAi) embryos was detected as gradual tightening of the CYK-4 peak width (arrows) after sudden widening (arrowheads). The grey arrows indicate the loss of CYK-4 accumulation in spd-1(RNAi) embryos and the central spindle-independent late recruitment. The mean and standard error were determined from 33, 7, 6 and 6 embryos for the control, efa-6(RNAi), bmk-1(RNAi) and spd-1(RNAi), respectively.
Mentions: To examine the response of the central spindle to increased pulling forces (Fig. 1a), we generated transgenic C. elegans strains expressing mCherry-tagged tubulin and green fluorescent protein (GFP)-tagged CYK-4. During the first mitotic division of control embryos, the bipolar spindle formed promptly following nuclear envelope breakdown (NEBD, time=0). NEBD was monitored by the loss of exclusion of the tubulin signal from pronuclei (Fig. 1b and Supplementary Fig. 1 for grayscale images covering the whole embryos). Approximately 120–150 s following NEBD, the distance between the two spindle poles (Fig. 1c) started to increase, indicating the onset of anaphase, as previously reported30 (Fig. 1d). At the same time, CYK-4 began to accumulate at the spindle midzone (Fig. 1b, control), where the plus ends of the two sets of interpolar microtubules from the two spindle poles form an interdigitating overlap, as confirmed by an accelerated increase in the peak intensity (Fig. 1d, control and Supplementary Fig. 2a for examples of the line profiling of CYK-4::GFP intensity). Within the next 30–60 s, the central spindle was established, with CYK-4 enriching rapidly in the central overlap zone and remaining there for ∼300 s until the cleavage furrow fully ingressed and the central spindle was compacted to form the midbody.

Bottom Line: The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis.How the cell balances these potentially conflicting requirements is poorly understood.This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

View Article: PubMed Central - PubMed

Affiliation: 1] Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK [2] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

ABSTRACT
During animal cell division, the central spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the central spindle is the only structure linking the two halves of the mitotic spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine spindle positioning and drive chromosome segregation through spindle elongation. The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the central spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

No MeSH data available.


Related in: MedlinePlus