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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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PRC1SPD-1depletion prevents the formation of midbody microtubule bundles and Aurora BAIR-2 targeting to the intercellular bridge. (A) Central plane confocal images of control (top; n = 8 embryos) and PRC1spd-1(RNAi) (bottom; n = 9) embryos expressing GFP–β-tubulin and mCherry-histone. Kymographs of the GFP–β-tubulin signal in the midbody region are also shown. Times are seconds after furrow initiation. (B) Central plane confocal images of control (n = 5) and PRC1spd-1(RNAi) (n = 5) embryos expressing GFP–Aurora BAIR-2 along with the mCherry-tagged plasma membrane probe. Times are seconds after furrow initiation. (C) The central region of confocal images of control (n = 18) and PRC1spd-1(RNAi) embryos (n = 7) expressing a GFP-tagged plasma membrane probe and mCherry-Mklp1ZEN-4 are shown at different time points after furrow initiation. (D) Deconvolved wide-field image (single z plane) of a control embryo stained for MyosinNMY-2 and MKLP1ZEN-4 along with tubulin and DNA (not depicted). DNA condensation and midbody compaction indicate that this is an abscission phase embryo whose furrow retracted during the freeze-crack fixation. Insets show MKLP1ZEN-4 at the midbody (white arrowhead) and overlapping with MyosinNMY-2 on the midbody ring. (E, top) Deconvolved wide-field images of control and PRC1spd-1(RNAi) embryos fixed during constriction phase (left) or abscission phase (right) and stained for tubulin (green), DNA, and Mklp1ZEN-4. Images are 2-µm projections through the central region of the embryo. (bottom) Traces show line scans (20 × 70 pixels) drawn across the center of control and PRC1spd-1(RNAi) embryos (n = 6 control and 6 PRC1spd-1(RNAi) embryos for each phase imaged in a single experiment). Intensity values were normalized by dividing by the mean fluorescence intensity in the cytoplasm near the cell periphery. a.u., arbitrary unit. White boxes on the low magnification images in B, D, and E mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
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fig3: PRC1SPD-1depletion prevents the formation of midbody microtubule bundles and Aurora BAIR-2 targeting to the intercellular bridge. (A) Central plane confocal images of control (top; n = 8 embryos) and PRC1spd-1(RNAi) (bottom; n = 9) embryos expressing GFP–β-tubulin and mCherry-histone. Kymographs of the GFP–β-tubulin signal in the midbody region are also shown. Times are seconds after furrow initiation. (B) Central plane confocal images of control (n = 5) and PRC1spd-1(RNAi) (n = 5) embryos expressing GFP–Aurora BAIR-2 along with the mCherry-tagged plasma membrane probe. Times are seconds after furrow initiation. (C) The central region of confocal images of control (n = 18) and PRC1spd-1(RNAi) embryos (n = 7) expressing a GFP-tagged plasma membrane probe and mCherry-Mklp1ZEN-4 are shown at different time points after furrow initiation. (D) Deconvolved wide-field image (single z plane) of a control embryo stained for MyosinNMY-2 and MKLP1ZEN-4 along with tubulin and DNA (not depicted). DNA condensation and midbody compaction indicate that this is an abscission phase embryo whose furrow retracted during the freeze-crack fixation. Insets show MKLP1ZEN-4 at the midbody (white arrowhead) and overlapping with MyosinNMY-2 on the midbody ring. (E, top) Deconvolved wide-field images of control and PRC1spd-1(RNAi) embryos fixed during constriction phase (left) or abscission phase (right) and stained for tubulin (green), DNA, and Mklp1ZEN-4. Images are 2-µm projections through the central region of the embryo. (bottom) Traces show line scans (20 × 70 pixels) drawn across the center of control and PRC1spd-1(RNAi) embryos (n = 6 control and 6 PRC1spd-1(RNAi) embryos for each phase imaged in a single experiment). Intensity values were normalized by dividing by the mean fluorescence intensity in the cytoplasm near the cell periphery. a.u., arbitrary unit. White boxes on the low magnification images in B, D, and E mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.

Mentions: To examine the role of midbody microtubules in abscission, we depleted the microtubule-bundling protein PRC1SPD-1. As in other systems (Mollinari et al., 2002, 2005; Vernì et al., 2004; D’Avino et al., 2007), PRC1SPD-1 inhibition in the C. elegans embryo prevents the formation of the microtubule bundles that make up the central spindle and blocks midbody assembly (Verbrugghe and White, 2004). We confirmed loss of midbody microtubules by imaging control and PRC1spd-1(RNAi) embryos expressing GFP–β-tubulin and mCherry::histone. In control embryos, bundled microtubules in the central spindle compacted to form the midbody, which could be monitored for >400 s after furrow initiation. After this point, which corresponds to the onset of mitosis of the second cell division, midbody microtubules appeared to dissipate, suggesting that relatively few microtubules span the intracellular bridge at the time of midbody release in control C. elegans embryos. In contrast, no central spindle or midbody microtubules were detected at any stage in PRC1spd-1(RNAi) embryos (Fig. 3 A and Video 7). Consistent with the absence of midbody microtubules, as the furrow closed during the first division, we could not detect any focus of GFP–Aurora BAIR-2 or mCherry-Mklp1ZEN-4 embedded within the cell–cell boundary in PRC1spd-1(RNAi) embryos (Fig. 3, B and C). Although mCherry-Mklp1ZEN-4 did not localize to the initial cell–cell boundary in PRC1spd-1(RNAi) embryos, a population of mCherry-Mklp1ZEN-4 was subsequently recruited to the midbody ring, becoming detectable ∼500–600 s after furrow initiation (Fig. 3 C). In control embryos, the amount of mCherry-Mklp1ZEN-4 in the focus at the cell–cell boundary also increased over time (Fig. 3 C). In fixed abscission stage embryos, the freeze-crack fixation procedure occasionally causes the midbody ring to release from the midbody; in such embryos, Mklp1ZEN-4 localized to both the midbody and with Myosin IINMY-2 to the midbody ring (Fig. 3 D). These findings are consistent with work in vertebrate cells, suggesting that Mklp1 transitions from the midbody to the midbody ring as the intercellular bridge matures (Elia et al., 2011; Hu et al., 2012). To further confirm the loss of midbody microtubules in PRC1spd-1(RNAi) embryos, we performed immunofluorescence in fixed embryos using Mlkp1ZEN-4 as a marker for the location of the midbody ring. Whereas an intense microtubule bundle passed through the Mklp1ZEN-4-marked midbody ring in interphase two-cell stage control embryos, no tubulin fluorescence above background was detected passing through the Mklp1ZEN-4-marked midbody ring in PRC1spd-1(RNAi) embryos (Fig. 3 E). We conclude that there are no detectable microtubule bundles passing through the intercellular bridge in PRC1spd-1(RNAi) embryos. Our results further suggest that Mklp1ZEN-4 is a component of the midbody ring as well as the midbody and can be directly recruited to the midbody ring independent of midbody microtubules.


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)

PRC1SPD-1depletion prevents the formation of midbody microtubule bundles and Aurora BAIR-2 targeting to the intercellular bridge. (A) Central plane confocal images of control (top; n = 8 embryos) and PRC1spd-1(RNAi) (bottom; n = 9) embryos expressing GFP–β-tubulin and mCherry-histone. Kymographs of the GFP–β-tubulin signal in the midbody region are also shown. Times are seconds after furrow initiation. (B) Central plane confocal images of control (n = 5) and PRC1spd-1(RNAi) (n = 5) embryos expressing GFP–Aurora BAIR-2 along with the mCherry-tagged plasma membrane probe. Times are seconds after furrow initiation. (C) The central region of confocal images of control (n = 18) and PRC1spd-1(RNAi) embryos (n = 7) expressing a GFP-tagged plasma membrane probe and mCherry-Mklp1ZEN-4 are shown at different time points after furrow initiation. (D) Deconvolved wide-field image (single z plane) of a control embryo stained for MyosinNMY-2 and MKLP1ZEN-4 along with tubulin and DNA (not depicted). DNA condensation and midbody compaction indicate that this is an abscission phase embryo whose furrow retracted during the freeze-crack fixation. Insets show MKLP1ZEN-4 at the midbody (white arrowhead) and overlapping with MyosinNMY-2 on the midbody ring. (E, top) Deconvolved wide-field images of control and PRC1spd-1(RNAi) embryos fixed during constriction phase (left) or abscission phase (right) and stained for tubulin (green), DNA, and Mklp1ZEN-4. Images are 2-µm projections through the central region of the embryo. (bottom) Traces show line scans (20 × 70 pixels) drawn across the center of control and PRC1spd-1(RNAi) embryos (n = 6 control and 6 PRC1spd-1(RNAi) embryos for each phase imaged in a single experiment). Intensity values were normalized by dividing by the mean fluorescence intensity in the cytoplasm near the cell periphery. a.u., arbitrary unit. White boxes on the low magnification images in B, D, and E mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
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Related In: Results  -  Collection

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Show All Figures
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fig3: PRC1SPD-1depletion prevents the formation of midbody microtubule bundles and Aurora BAIR-2 targeting to the intercellular bridge. (A) Central plane confocal images of control (top; n = 8 embryos) and PRC1spd-1(RNAi) (bottom; n = 9) embryos expressing GFP–β-tubulin and mCherry-histone. Kymographs of the GFP–β-tubulin signal in the midbody region are also shown. Times are seconds after furrow initiation. (B) Central plane confocal images of control (n = 5) and PRC1spd-1(RNAi) (n = 5) embryos expressing GFP–Aurora BAIR-2 along with the mCherry-tagged plasma membrane probe. Times are seconds after furrow initiation. (C) The central region of confocal images of control (n = 18) and PRC1spd-1(RNAi) embryos (n = 7) expressing a GFP-tagged plasma membrane probe and mCherry-Mklp1ZEN-4 are shown at different time points after furrow initiation. (D) Deconvolved wide-field image (single z plane) of a control embryo stained for MyosinNMY-2 and MKLP1ZEN-4 along with tubulin and DNA (not depicted). DNA condensation and midbody compaction indicate that this is an abscission phase embryo whose furrow retracted during the freeze-crack fixation. Insets show MKLP1ZEN-4 at the midbody (white arrowhead) and overlapping with MyosinNMY-2 on the midbody ring. (E, top) Deconvolved wide-field images of control and PRC1spd-1(RNAi) embryos fixed during constriction phase (left) or abscission phase (right) and stained for tubulin (green), DNA, and Mklp1ZEN-4. Images are 2-µm projections through the central region of the embryo. (bottom) Traces show line scans (20 × 70 pixels) drawn across the center of control and PRC1spd-1(RNAi) embryos (n = 6 control and 6 PRC1spd-1(RNAi) embryos for each phase imaged in a single experiment). Intensity values were normalized by dividing by the mean fluorescence intensity in the cytoplasm near the cell periphery. a.u., arbitrary unit. White boxes on the low magnification images in B, D, and E mark the location of the region shown at higher magnification in the images at the bottom. Bars, 5 µm.
Mentions: To examine the role of midbody microtubules in abscission, we depleted the microtubule-bundling protein PRC1SPD-1. As in other systems (Mollinari et al., 2002, 2005; Vernì et al., 2004; D’Avino et al., 2007), PRC1SPD-1 inhibition in the C. elegans embryo prevents the formation of the microtubule bundles that make up the central spindle and blocks midbody assembly (Verbrugghe and White, 2004). We confirmed loss of midbody microtubules by imaging control and PRC1spd-1(RNAi) embryos expressing GFP–β-tubulin and mCherry::histone. In control embryos, bundled microtubules in the central spindle compacted to form the midbody, which could be monitored for >400 s after furrow initiation. After this point, which corresponds to the onset of mitosis of the second cell division, midbody microtubules appeared to dissipate, suggesting that relatively few microtubules span the intracellular bridge at the time of midbody release in control C. elegans embryos. In contrast, no central spindle or midbody microtubules were detected at any stage in PRC1spd-1(RNAi) embryos (Fig. 3 A and Video 7). Consistent with the absence of midbody microtubules, as the furrow closed during the first division, we could not detect any focus of GFP–Aurora BAIR-2 or mCherry-Mklp1ZEN-4 embedded within the cell–cell boundary in PRC1spd-1(RNAi) embryos (Fig. 3, B and C). Although mCherry-Mklp1ZEN-4 did not localize to the initial cell–cell boundary in PRC1spd-1(RNAi) embryos, a population of mCherry-Mklp1ZEN-4 was subsequently recruited to the midbody ring, becoming detectable ∼500–600 s after furrow initiation (Fig. 3 C). In control embryos, the amount of mCherry-Mklp1ZEN-4 in the focus at the cell–cell boundary also increased over time (Fig. 3 C). In fixed abscission stage embryos, the freeze-crack fixation procedure occasionally causes the midbody ring to release from the midbody; in such embryos, Mklp1ZEN-4 localized to both the midbody and with Myosin IINMY-2 to the midbody ring (Fig. 3 D). These findings are consistent with work in vertebrate cells, suggesting that Mklp1 transitions from the midbody to the midbody ring as the intercellular bridge matures (Elia et al., 2011; Hu et al., 2012). To further confirm the loss of midbody microtubules in PRC1spd-1(RNAi) embryos, we performed immunofluorescence in fixed embryos using Mlkp1ZEN-4 as a marker for the location of the midbody ring. Whereas an intense microtubule bundle passed through the Mklp1ZEN-4-marked midbody ring in interphase two-cell stage control embryos, no tubulin fluorescence above background was detected passing through the Mklp1ZEN-4-marked midbody ring in PRC1spd-1(RNAi) embryos (Fig. 3 E). We conclude that there are no detectable microtubule bundles passing through the intercellular bridge in PRC1spd-1(RNAi) embryos. Our results further suggest that Mklp1ZEN-4 is a component of the midbody ring as well as the midbody and can be directly recruited to the midbody ring independent of midbody microtubules.

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