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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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Furrow ingression and cytoplasmic isolation occur with normal timing in the absence of midbody microtubules. (A) Graph plotting mean furrow diameter versus time, for the first division of control (reproduced from Fig. 1 A for comparison) and PRC1spd-1(RNAi) embryos (n = 10 embryos for each condition). Error bars are the SDs. Arrow denotes the last time point with a measurable opening (apparent closure). (B and C) Central plane confocal images of control and PRC1spd-1(RNAi) embryos expressing Myosin IINMY-2–GFP along with the mCherry-tagged plasma membrane probe (B, n = 5 for each condition) or GFP-septinUNC-59 along with the mCherry-tagged plasma membrane probe and mCherry-histone (C; n = 5 for each condition). Times are seconds after furrow initiation. (D) Outline of the method used to compare cytoplasmic isolation kinetics in control and PRC1spd-1(RNAi) embryos. Embryos expressing the GFP-tagged plasma membrane probe loaded with 10-kD caged carboxy-Q-rhodamine–labeled dextran were photoactivated on one side at different time points after furrow initiation, and images were collected at 5-s intervals to monitor probe diffusion. Kymographs are shown for embryos photoactivated early in cytokinesis, midcytokinesis, and at closure (the early and closure kymographs are reproduced from Fig. 1 B). Red arrows denote the point of photoactivation. The NID between the activated (A) and unactivated (U) halves of the embryo, calculated as shown, was plotted versus time, and the initial slope of the intensity difference, which reflects the rate of diffusion across the division plane, was calculated. (E) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and PRC1spd-1(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. White boxes on the low magnification images in B and C mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
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fig4: Furrow ingression and cytoplasmic isolation occur with normal timing in the absence of midbody microtubules. (A) Graph plotting mean furrow diameter versus time, for the first division of control (reproduced from Fig. 1 A for comparison) and PRC1spd-1(RNAi) embryos (n = 10 embryos for each condition). Error bars are the SDs. Arrow denotes the last time point with a measurable opening (apparent closure). (B and C) Central plane confocal images of control and PRC1spd-1(RNAi) embryos expressing Myosin IINMY-2–GFP along with the mCherry-tagged plasma membrane probe (B, n = 5 for each condition) or GFP-septinUNC-59 along with the mCherry-tagged plasma membrane probe and mCherry-histone (C; n = 5 for each condition). Times are seconds after furrow initiation. (D) Outline of the method used to compare cytoplasmic isolation kinetics in control and PRC1spd-1(RNAi) embryos. Embryos expressing the GFP-tagged plasma membrane probe loaded with 10-kD caged carboxy-Q-rhodamine–labeled dextran were photoactivated on one side at different time points after furrow initiation, and images were collected at 5-s intervals to monitor probe diffusion. Kymographs are shown for embryos photoactivated early in cytokinesis, midcytokinesis, and at closure (the early and closure kymographs are reproduced from Fig. 1 B). Red arrows denote the point of photoactivation. The NID between the activated (A) and unactivated (U) halves of the embryo, calculated as shown, was plotted versus time, and the initial slope of the intensity difference, which reflects the rate of diffusion across the division plane, was calculated. (E) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and PRC1spd-1(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. White boxes on the low magnification images in B and C mark the location of the region shown at higher magnification in the images at the bottom. 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)

Furrow ingression and cytoplasmic isolation occur with normal timing in the absence of midbody microtubules. (A) Graph plotting mean furrow diameter versus time, for the first division of control (reproduced from Fig. 1 A for comparison) and PRC1spd-1(RNAi) embryos (n = 10 embryos for each condition). Error bars are the SDs. Arrow denotes the last time point with a measurable opening (apparent closure). (B and C) Central plane confocal images of control and PRC1spd-1(RNAi) embryos expressing Myosin IINMY-2–GFP along with the mCherry-tagged plasma membrane probe (B, n = 5 for each condition) or GFP-septinUNC-59 along with the mCherry-tagged plasma membrane probe and mCherry-histone (C; n = 5 for each condition). Times are seconds after furrow initiation. (D) Outline of the method used to compare cytoplasmic isolation kinetics in control and PRC1spd-1(RNAi) embryos. Embryos expressing the GFP-tagged plasma membrane probe loaded with 10-kD caged carboxy-Q-rhodamine–labeled dextran were photoactivated on one side at different time points after furrow initiation, and images were collected at 5-s intervals to monitor probe diffusion. Kymographs are shown for embryos photoactivated early in cytokinesis, midcytokinesis, and at closure (the early and closure kymographs are reproduced from Fig. 1 B). Red arrows denote the point of photoactivation. The NID between the activated (A) and unactivated (U) halves of the embryo, calculated as shown, was plotted versus time, and the initial slope of the intensity difference, which reflects the rate of diffusion across the division plane, was calculated. (E) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and PRC1spd-1(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. White boxes on the low magnification images in B and C mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824018&req=5

fig4: Furrow ingression and cytoplasmic isolation occur with normal timing in the absence of midbody microtubules. (A) Graph plotting mean furrow diameter versus time, for the first division of control (reproduced from Fig. 1 A for comparison) and PRC1spd-1(RNAi) embryos (n = 10 embryos for each condition). Error bars are the SDs. Arrow denotes the last time point with a measurable opening (apparent closure). (B and C) Central plane confocal images of control and PRC1spd-1(RNAi) embryos expressing Myosin IINMY-2–GFP along with the mCherry-tagged plasma membrane probe (B, n = 5 for each condition) or GFP-septinUNC-59 along with the mCherry-tagged plasma membrane probe and mCherry-histone (C; n = 5 for each condition). Times are seconds after furrow initiation. (D) Outline of the method used to compare cytoplasmic isolation kinetics in control and PRC1spd-1(RNAi) embryos. Embryos expressing the GFP-tagged plasma membrane probe loaded with 10-kD caged carboxy-Q-rhodamine–labeled dextran were photoactivated on one side at different time points after furrow initiation, and images were collected at 5-s intervals to monitor probe diffusion. Kymographs are shown for embryos photoactivated early in cytokinesis, midcytokinesis, and at closure (the early and closure kymographs are reproduced from Fig. 1 B). Red arrows denote the point of photoactivation. The NID between the activated (A) and unactivated (U) halves of the embryo, calculated as shown, was plotted versus time, and the initial slope of the intensity difference, which reflects the rate of diffusion across the division plane, was calculated. (E) Graph plotting the mean initial slope of the NID versus time in seconds after furrow initiation for control and PRC1spd-1(RNAi) embryos. Error bars are the 90% confidence interval; mean n = 10 slope measurements per time point. White boxes on the low magnification images in B and C mark the location of the region shown at higher magnification in the images at the bottom. 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