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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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Midbody microtubules are not required for membrane shedding, ESCRT recruitment, or midbody ring release. (A) Central plane confocal images showing membrane shedding (white arrowheads) at the cell–cell boundary in a PRC1spd-1(RNAi) embryo (n = 9 embryos) expressing an mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. Times are relative to anaphase of the second division. (B) Central plane confocal images showing midbody ring release in a PRC1spd-1(RNAi) embryo expressing the midbody ring markers Myosin IINMY-2–GFP (n = 8 embryos), GFP–CYK-7 (n = 10 embryos), or GFP-septinUNC-59 (n = 5 embryos) along with the mCherry-tagged plasma membrane probe and mCherry-histone. Times are relative to anaphase of the second division. Midbody rings are highlighted before (yellow arrows) and after (green arrows) release from the cell–cell junction. (C) Graphs plotting the mean onset of membrane shedding (top) and midbody ring release (bottom) for control and PRC1spd-1(RNAi) embryos. Error bars are the SDs. (D) Central plane confocal images of control (n = 6 embryos) and PRC1spd-1(RNAi) (n = 7 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Images are scaled equivalently. White boxes on the low magnification images in A and B mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
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fig5: Midbody microtubules are not required for membrane shedding, ESCRT recruitment, or midbody ring release. (A) Central plane confocal images showing membrane shedding (white arrowheads) at the cell–cell boundary in a PRC1spd-1(RNAi) embryo (n = 9 embryos) expressing an mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. Times are relative to anaphase of the second division. (B) Central plane confocal images showing midbody ring release in a PRC1spd-1(RNAi) embryo expressing the midbody ring markers Myosin IINMY-2–GFP (n = 8 embryos), GFP–CYK-7 (n = 10 embryos), or GFP-septinUNC-59 (n = 5 embryos) along with the mCherry-tagged plasma membrane probe and mCherry-histone. Times are relative to anaphase of the second division. Midbody rings are highlighted before (yellow arrows) and after (green arrows) release from the cell–cell junction. (C) Graphs plotting the mean onset of membrane shedding (top) and midbody ring release (bottom) for control and PRC1spd-1(RNAi) embryos. Error bars are the SDs. (D) Central plane confocal images of control (n = 6 embryos) and PRC1spd-1(RNAi) (n = 7 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Images are scaled equivalently. White boxes on the low magnification images in A and B mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.

Mentions: Next, we monitored abscission in PRC1spd-1(RNAi) embryos. The kinetics of contractile ring closure in PRC1spd-1(RNAi) embryos were similar to those in controls, and apparent closure of the hole between the daughter cells occurred at a similar time point (Fig. 4 A). Monitoring of the contractile/midbody ring components Myosin IINMY-2–GFP, GFP-SeptinUNC-59, and GFP–CYK-7 revealed that despite the absence of the midzone/midbody, the contractile ring closed and was converted into a midbody ring embedded in the cell–cell boundary with normal kinetics in PRC1spd-1(RNAi) embryos (Fig. 4, B and C; and Fig. 5 B).


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)

Midbody microtubules are not required for membrane shedding, ESCRT recruitment, or midbody ring release. (A) Central plane confocal images showing membrane shedding (white arrowheads) at the cell–cell boundary in a PRC1spd-1(RNAi) embryo (n = 9 embryos) expressing an mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. Times are relative to anaphase of the second division. (B) Central plane confocal images showing midbody ring release in a PRC1spd-1(RNAi) embryo expressing the midbody ring markers Myosin IINMY-2–GFP (n = 8 embryos), GFP–CYK-7 (n = 10 embryos), or GFP-septinUNC-59 (n = 5 embryos) along with the mCherry-tagged plasma membrane probe and mCherry-histone. Times are relative to anaphase of the second division. Midbody rings are highlighted before (yellow arrows) and after (green arrows) release from the cell–cell junction. (C) Graphs plotting the mean onset of membrane shedding (top) and midbody ring release (bottom) for control and PRC1spd-1(RNAi) embryos. Error bars are the SDs. (D) Central plane confocal images of control (n = 6 embryos) and PRC1spd-1(RNAi) (n = 7 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Images are scaled equivalently. White boxes on the low magnification images in A and B mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
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Related In: Results  -  Collection

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fig5: Midbody microtubules are not required for membrane shedding, ESCRT recruitment, or midbody ring release. (A) Central plane confocal images showing membrane shedding (white arrowheads) at the cell–cell boundary in a PRC1spd-1(RNAi) embryo (n = 9 embryos) expressing an mCherry-tagged plasma membrane probe and GFP–Aurora BAIR-2. Times are relative to anaphase of the second division. (B) Central plane confocal images showing midbody ring release in a PRC1spd-1(RNAi) embryo expressing the midbody ring markers Myosin IINMY-2–GFP (n = 8 embryos), GFP–CYK-7 (n = 10 embryos), or GFP-septinUNC-59 (n = 5 embryos) along with the mCherry-tagged plasma membrane probe and mCherry-histone. Times are relative to anaphase of the second division. Midbody rings are highlighted before (yellow arrows) and after (green arrows) release from the cell–cell junction. (C) Graphs plotting the mean onset of membrane shedding (top) and midbody ring release (bottom) for control and PRC1spd-1(RNAi) embryos. Error bars are the SDs. (D) Central plane confocal images of control (n = 6 embryos) and PRC1spd-1(RNAi) (n = 7 embryos) embryos expressing GFP–ESCRT-IMVB-12. Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. Images are scaled equivalently. White boxes on the low magnification images in A and B mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
Mentions: Next, we monitored abscission in PRC1spd-1(RNAi) embryos. The kinetics of contractile ring closure in PRC1spd-1(RNAi) embryos were similar to those in controls, and apparent closure of the hole between the daughter cells occurred at a similar time point (Fig. 4 A). Monitoring of the contractile/midbody ring components Myosin IINMY-2–GFP, GFP-SeptinUNC-59, and GFP–CYK-7 revealed that despite the absence of the midzone/midbody, the contractile ring closed and was converted into a midbody ring embedded in the cell–cell boundary with normal kinetics in PRC1spd-1(RNAi) embryos (Fig. 4, B and C; and Fig. 5 B).

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