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The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules.

Green RA, Mayers JR, Wang S, Lewellyn L, Desai A, Audhya A, Oegema K - J. Cell Biol. (2013)

Bottom Line: Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery.Surprisingly, midbody microtubules were dispensable for both stages.These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093.

ABSTRACT
Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring-derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

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The septins are required for timely cytoplasmic isolation and for midbody release. (A) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and septinunc-59(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. (B and C, top) Central plane confocal images of control and septinunc-59(RNAi) embryos expressing a fluorescently tagged plasma membrane probe and the midbody markers mCherry-Mklp1ZEN-4 (B; n = 11 embryos) or GFP–CYK-7 (C; n = 11 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe are indicated (white arrowheads). Arrows point to the midbody/midbody ring from the first division, which is released in control embryos (green arrows) and fails to be released in septinunc-59(RNAi) embryos (yellow arrows). Asterisks mark the tip of the ingressing furrow from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released. In cases in which the midbody/midbody ring was not released, the data point refers to the endpoint of the time-lapse sequence. (D) The central region of confocal images of control (n = 11) and septinunc-59(RNAi) (n = 10) embryos expressing the mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. (E) Confocal images of septinunc-59(RNAi) (n = 6 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times in D and E are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Bars, 5 µm.
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fig6: The septins are required for timely cytoplasmic isolation and for midbody release. (A) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and septinunc-59(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. (B and C, top) Central plane confocal images of control and septinunc-59(RNAi) embryos expressing a fluorescently tagged plasma membrane probe and the midbody markers mCherry-Mklp1ZEN-4 (B; n = 11 embryos) or GFP–CYK-7 (C; n = 11 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe are indicated (white arrowheads). Arrows point to the midbody/midbody ring from the first division, which is released in control embryos (green arrows) and fails to be released in septinunc-59(RNAi) embryos (yellow arrows). Asterisks mark the tip of the ingressing furrow from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released. In cases in which the midbody/midbody ring was not released, the data point refers to the endpoint of the time-lapse sequence. (D) The central region of confocal images of control (n = 11) and septinunc-59(RNAi) (n = 10) embryos expressing the mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. (E) Confocal images of septinunc-59(RNAi) (n = 6 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times in D and E are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Bars, 5 µm.

Mentions: The septins are midbody ring components across metazoans and have been shown to be important for abscission in vertebrate and Drosophila S2 cells (Estey et al., 2010; Kechad et al., 2012). In contrast to the combinatorial complexity of hetero-oligomeric septin complexes in humans, which have 13 different septins (Hall and Russell, 2012), C. elegans has only two septins (UNC-59 and UNC-61), and depletion of either is sufficient to disrupt septin recruitment to the contractile ring (Nguyen et al., 2000; John et al., 2007). To examine the effects of septin inhibition, we depleted the septin UNC-59. Consistent with previous work (Maddox et al., 2007), the furrow closed with similar kinetics to controls until the very end, when septinunc-59(RNAi) embryos persisted longer (∼40 s) with a small hole between the daughter cells (Fig. S4 A). To determine how septin depletion affects the timing of cytoplasmic isolation, we used the approach described in Fig. 4 D to monitor the diffusion of photoactivated 10-kD dextran across the division plane. Plotting the mean initial slope of the NID versus time revealed that the rate of diffusion across the division plane decreases with similar kinetics for the first 250 s after furrow initiation. However, at this point, the curves diverge, and the septinunc-59(RNAi) embryos remain diffusionally connected, with a small open channel between the daughter cells, for ∼140 s longer than controls (Fig. 6 A). We conclude that cytoplasmic isolation is substantially delayed by septin depletion.


The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules.

Green RA, Mayers JR, Wang S, Lewellyn L, Desai A, Audhya A, Oegema K - J. Cell Biol. (2013)

The septins are required for timely cytoplasmic isolation and for midbody release. (A) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and septinunc-59(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. (B and C, top) Central plane confocal images of control and septinunc-59(RNAi) embryos expressing a fluorescently tagged plasma membrane probe and the midbody markers mCherry-Mklp1ZEN-4 (B; n = 11 embryos) or GFP–CYK-7 (C; n = 11 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe are indicated (white arrowheads). Arrows point to the midbody/midbody ring from the first division, which is released in control embryos (green arrows) and fails to be released in septinunc-59(RNAi) embryos (yellow arrows). Asterisks mark the tip of the ingressing furrow from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released. In cases in which the midbody/midbody ring was not released, the data point refers to the endpoint of the time-lapse sequence. (D) The central region of confocal images of control (n = 11) and septinunc-59(RNAi) (n = 10) embryos expressing the mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. (E) Confocal images of septinunc-59(RNAi) (n = 6 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times in D and E are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig6: The septins are required for timely cytoplasmic isolation and for midbody release. (A) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and septinunc-59(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. (B and C, top) Central plane confocal images of control and septinunc-59(RNAi) embryos expressing a fluorescently tagged plasma membrane probe and the midbody markers mCherry-Mklp1ZEN-4 (B; n = 11 embryos) or GFP–CYK-7 (C; n = 11 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe are indicated (white arrowheads). Arrows point to the midbody/midbody ring from the first division, which is released in control embryos (green arrows) and fails to be released in septinunc-59(RNAi) embryos (yellow arrows). Asterisks mark the tip of the ingressing furrow from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released. In cases in which the midbody/midbody ring was not released, the data point refers to the endpoint of the time-lapse sequence. (D) The central region of confocal images of control (n = 11) and septinunc-59(RNAi) (n = 10) embryos expressing the mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. (E) Confocal images of septinunc-59(RNAi) (n = 6 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times in D and E are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Bars, 5 µm.
Mentions: The septins are midbody ring components across metazoans and have been shown to be important for abscission in vertebrate and Drosophila S2 cells (Estey et al., 2010; Kechad et al., 2012). In contrast to the combinatorial complexity of hetero-oligomeric septin complexes in humans, which have 13 different septins (Hall and Russell, 2012), C. elegans has only two septins (UNC-59 and UNC-61), and depletion of either is sufficient to disrupt septin recruitment to the contractile ring (Nguyen et al., 2000; John et al., 2007). To examine the effects of septin inhibition, we depleted the septin UNC-59. Consistent with previous work (Maddox et al., 2007), the furrow closed with similar kinetics to controls until the very end, when septinunc-59(RNAi) embryos persisted longer (∼40 s) with a small hole between the daughter cells (Fig. S4 A). To determine how septin depletion affects the timing of cytoplasmic isolation, we used the approach described in Fig. 4 D to monitor the diffusion of photoactivated 10-kD dextran across the division plane. Plotting the mean initial slope of the NID versus time revealed that the rate of diffusion across the division plane decreases with similar kinetics for the first 250 s after furrow initiation. However, at this point, the curves diverge, and the septinunc-59(RNAi) embryos remain diffusionally connected, with a small open channel between the daughter cells, for ∼140 s longer than controls (Fig. 6 A). We conclude that cytoplasmic isolation is substantially delayed by septin depletion.

Bottom Line: Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery.Surprisingly, midbody microtubules were dispensable for both stages.These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093.

ABSTRACT
Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring-derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

Show MeSH
Related in: MedlinePlus