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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 ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising 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 control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
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fig2: The ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising 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 control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.

Mentions: These results suggest that abscission in the early C. elegans embryo occurs in two stages (Fig. 2 F). During the first stage, the cytoplasm in the two daughter cells becomes diffusionally isolated, coincident with the completion of furrowing and formation of the intercellular bridge. During the second stage, which begins during anaphase of the subsequent cell division, the cortex surrounding the midbody is remodeled, releasing fragments containing plasma membrane and midbody ring markers. Remodeling culminates, ∼200 s later, in the release of the midbody and midbody ring into the posterior cell.


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 ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising 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 control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig2: The ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising 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 control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
Mentions: These results suggest that abscission in the early C. elegans embryo occurs in two stages (Fig. 2 F). During the first stage, the cytoplasm in the two daughter cells becomes diffusionally isolated, coincident with the completion of furrowing and formation of the intercellular bridge. During the second stage, which begins during anaphase of the subsequent cell division, the cortex surrounding the midbody is remodeled, releasing fragments containing plasma membrane and midbody ring markers. Remodeling culminates, ∼200 s later, in the release of the midbody and midbody ring into the posterior cell.

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