Limits...
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.

Show MeSH

Related in: MedlinePlus

The septins and ESCRT machinery function at different stages of abscission. (A, left) The central region of confocal images of control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) (>10 embryos for each condition) embryos expressing a GFP-tagged plasma membrane probe at different time points after furrow initiation. (right) Differential interference contrast (DIC) images of control, septinunc-59(RNAi), or ESCRT-Itsg-101(RNAi) embryos at the two-cell stage. The region in the yellow boxes is shown at higher magnification in the images to the right. (B) Central plane confocal images of four-cell stage control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-MKLP1ZEN-4 (top; n = 21 control, 12 septinunc-59(RNAi), and 16 ESCRT-Itsg-101(RNAi) embryos) or the midbody ring marker GFP–CYK-7 (bottom; n = 19 control, 12 septinunc-59(RNAi), and 6 ESCRT-Itsg-101(RNAi) embryos). The midbody is released into the posterior cell in control embryos, protrudes from cell–cell boundary in septinunc-59(RNAi) embryos, and is enclosed within a plasma membrane–bound compartment embedded in the cell–cell boundary in ESCRT-Itsg-101(RNAi) embryos. White boxes on the low magnification images mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: The septins and ESCRT machinery function at different stages of abscission. (A, left) The central region of confocal images of control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) (>10 embryos for each condition) embryos expressing a GFP-tagged plasma membrane probe at different time points after furrow initiation. (right) Differential interference contrast (DIC) images of control, septinunc-59(RNAi), or ESCRT-Itsg-101(RNAi) embryos at the two-cell stage. The region in the yellow boxes is shown at higher magnification in the images to the right. (B) Central plane confocal images of four-cell stage control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-MKLP1ZEN-4 (top; n = 21 control, 12 septinunc-59(RNAi), and 16 ESCRT-Itsg-101(RNAi) embryos) or the midbody ring marker GFP–CYK-7 (bottom; n = 19 control, 12 septinunc-59(RNAi), and 6 ESCRT-Itsg-101(RNAi) embryos). The midbody is released into the posterior cell in control embryos, protrudes from cell–cell boundary in septinunc-59(RNAi) embryos, and is enclosed within a plasma membrane–bound compartment embedded in the cell–cell boundary in ESCRT-Itsg-101(RNAi) embryos. White boxes on the low magnification images mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.

Mentions: Because both ESCRT and septin inhibitions result in failure of midbody/midbody ring release (Fig. 2, D and E; and Fig. 6, B and C), we performed a more careful comparison of these two conditions. In control embryos, expressing a GFP-tagged plasma membrane probe, the ingressing furrow enveloped the midbody, generating a smooth cell–cell boundary. In septinunc-59(RNAi) embryos, envelopment of the midbody by the plasma membrane was delayed, but the boundary remained smooth (Fig. 6 A). In ESCRT-Itsg-101(RNAi) embryos, the furrow enveloped the midbody with normal timing, consistent with our analysis indicating that cytoplasmic isolation occurs coincident with the completion of furrowing (Fig. 2 C); however, the intercellular bridge was often distended, suggesting the presence of an obstruction enveloped along with the midbody (Fig. 7 A and Video 10). An occlusion was also visible in the cell–cell boundary in differential interference contrast images of ESCRT-Itsg-101(RNAi) embryos (Fig. 7 A). Given that we do not observe ESCRT-I on the midbody/midbody ring until after cytoplasmic isolation, we suspect that the obstruction is a consequence of the effect of ESCRT inhibition on the formation of multivesicular bodies (Henne et al., 2011; McCullough et al., 2013), rather than caused by its role in midbody/midbody release. The midbody release defect in septinUNC-59-depleted embryos also differed from that in ESCRT-ITSG-101–depleted embryos. In septinUNC-59-depleted embryos, the midbody/midbody ring protruded into the posterior cell and did not appear to be encased in plasma membrane marker (Fig. 7 B and Fig. 6, B and C). In contrast, in ESCRT-ITSG-101–depleted embryos, the midbody/midbody ring was encased in a ring of plasma membrane embedded in the cell–cell boundary (Fig. 7 B). These distinct defects suggest that the septins and the ESCRT machinery function at different points during abscission (Fig. 8 and accompanying text in the Discussion).


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 and ESCRT machinery function at different stages of abscission. (A, left) The central region of confocal images of control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) (>10 embryos for each condition) embryos expressing a GFP-tagged plasma membrane probe at different time points after furrow initiation. (right) Differential interference contrast (DIC) images of control, septinunc-59(RNAi), or ESCRT-Itsg-101(RNAi) embryos at the two-cell stage. The region in the yellow boxes is shown at higher magnification in the images to the right. (B) Central plane confocal images of four-cell stage control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-MKLP1ZEN-4 (top; n = 21 control, 12 septinunc-59(RNAi), and 16 ESCRT-Itsg-101(RNAi) embryos) or the midbody ring marker GFP–CYK-7 (bottom; n = 19 control, 12 septinunc-59(RNAi), and 6 ESCRT-Itsg-101(RNAi) embryos). The midbody is released into the posterior cell in control embryos, protrudes from cell–cell boundary in septinunc-59(RNAi) embryos, and is enclosed within a plasma membrane–bound compartment embedded in the cell–cell boundary in ESCRT-Itsg-101(RNAi) embryos. White boxes on the low magnification images mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: The septins and ESCRT machinery function at different stages of abscission. (A, left) The central region of confocal images of control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) (>10 embryos for each condition) embryos expressing a GFP-tagged plasma membrane probe at different time points after furrow initiation. (right) Differential interference contrast (DIC) images of control, septinunc-59(RNAi), or ESCRT-Itsg-101(RNAi) embryos at the two-cell stage. The region in the yellow boxes is shown at higher magnification in the images to the right. (B) Central plane confocal images of four-cell stage control, septinunc-59(RNAi), and ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-MKLP1ZEN-4 (top; n = 21 control, 12 septinunc-59(RNAi), and 16 ESCRT-Itsg-101(RNAi) embryos) or the midbody ring marker GFP–CYK-7 (bottom; n = 19 control, 12 septinunc-59(RNAi), and 6 ESCRT-Itsg-101(RNAi) embryos). The midbody is released into the posterior cell in control embryos, protrudes from cell–cell boundary in septinunc-59(RNAi) embryos, and is enclosed within a plasma membrane–bound compartment embedded in the cell–cell boundary in ESCRT-Itsg-101(RNAi) embryos. White boxes on the low magnification images mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.
Mentions: Because both ESCRT and septin inhibitions result in failure of midbody/midbody ring release (Fig. 2, D and E; and Fig. 6, B and C), we performed a more careful comparison of these two conditions. In control embryos, expressing a GFP-tagged plasma membrane probe, the ingressing furrow enveloped the midbody, generating a smooth cell–cell boundary. In septinunc-59(RNAi) embryos, envelopment of the midbody by the plasma membrane was delayed, but the boundary remained smooth (Fig. 6 A). In ESCRT-Itsg-101(RNAi) embryos, the furrow enveloped the midbody with normal timing, consistent with our analysis indicating that cytoplasmic isolation occurs coincident with the completion of furrowing (Fig. 2 C); however, the intercellular bridge was often distended, suggesting the presence of an obstruction enveloped along with the midbody (Fig. 7 A and Video 10). An occlusion was also visible in the cell–cell boundary in differential interference contrast images of ESCRT-Itsg-101(RNAi) embryos (Fig. 7 A). Given that we do not observe ESCRT-I on the midbody/midbody ring until after cytoplasmic isolation, we suspect that the obstruction is a consequence of the effect of ESCRT inhibition on the formation of multivesicular bodies (Henne et al., 2011; McCullough et al., 2013), rather than caused by its role in midbody/midbody release. The midbody release defect in septinUNC-59-depleted embryos also differed from that in ESCRT-ITSG-101–depleted embryos. In septinUNC-59-depleted embryos, the midbody/midbody ring protruded into the posterior cell and did not appear to be encased in plasma membrane marker (Fig. 7 B and Fig. 6, B and C). In contrast, in ESCRT-ITSG-101–depleted embryos, the midbody/midbody ring was encased in a ring of plasma membrane embedded in the cell–cell boundary (Fig. 7 B). These distinct defects suggest that the septins and the ESCRT machinery function at different points during abscission (Fig. 8 and accompanying text in the Discussion).

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