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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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Abscission occurs in two stages: cytoplasmic isolation and release of the midbody/midbody ring. (A) Furrow diameter was measured in projections of the central region of z stacks of embryos (n = 10) expressing a GFP-tagged plasma membrane probe. (right) Graph plots mean furrow diameter versus time after furrow initiation. Arrow indicates the last time point when a hole can be detected (apparent closure). Error bars are the SDs. (top) Schematics illustrate shape changes during the first division in the C. elegans embryo, highlighting intercellular bridge structure. MTs, microtubules. (B, left) Schematic illustrates the method for monitoring the diffusion of photoactivated dextran between the two half-cells before and after cytokinesis. Examples of probe diffusion in embryos photoactivated before cytokinesis onset (middle; n = 12 embryos) and after apparent closure (right; n = 8 embryos, example shown is 350 s after furrow initiation). Central plane images show the embryos before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). Kymographs were constructed by aligning strips (narrow rectangles) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (C) Central plane fluorescence confocal images of embryos expressing a fluorescently tagged plasma membrane probe (red in merged images) along with either the midbody marker mCherry-Mklp1ZEN-4 (n = 18 embryos) or the midbody ring marker Myosin IINMY-2–GFP (n = 14 embryos). Times are relative to anaphase of the second division (∼900 s after initiation of the first division furrow). Different embryos are shown to illustrate membrane shedding (−80 to 140–s time points) and midbody/midbody ring release (220–260-s time points). White boxes on the low magnification images mark the location of the region shown at higher magnification in the three adjacent panels. Loops and released fragments marked with the plasma membrane probe are indicated (white arrowheads). Yellow arrows denote mCherry-Mklp1ZEN-4–marked or Myosin IINMY-2–GFP-marked midbody remnants before release from the cell–cell junction. Green arrows mark the same components after release. Schematics illustrate events at each stage. Bars, 5 µm.
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fig1: Abscission occurs in two stages: cytoplasmic isolation and release of the midbody/midbody ring. (A) Furrow diameter was measured in projections of the central region of z stacks of embryos (n = 10) expressing a GFP-tagged plasma membrane probe. (right) Graph plots mean furrow diameter versus time after furrow initiation. Arrow indicates the last time point when a hole can be detected (apparent closure). Error bars are the SDs. (top) Schematics illustrate shape changes during the first division in the C. elegans embryo, highlighting intercellular bridge structure. MTs, microtubules. (B, left) Schematic illustrates the method for monitoring the diffusion of photoactivated dextran between the two half-cells before and after cytokinesis. Examples of probe diffusion in embryos photoactivated before cytokinesis onset (middle; n = 12 embryos) and after apparent closure (right; n = 8 embryos, example shown is 350 s after furrow initiation). Central plane images show the embryos before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). Kymographs were constructed by aligning strips (narrow rectangles) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (C) Central plane fluorescence confocal images of embryos expressing a fluorescently tagged plasma membrane probe (red in merged images) along with either the midbody marker mCherry-Mklp1ZEN-4 (n = 18 embryos) or the midbody ring marker Myosin IINMY-2–GFP (n = 14 embryos). Times are relative to anaphase of the second division (∼900 s after initiation of the first division furrow). Different embryos are shown to illustrate membrane shedding (−80 to 140–s time points) and midbody/midbody ring release (220–260-s time points). White boxes on the low magnification images mark the location of the region shown at higher magnification in the three adjacent panels. Loops and released fragments marked with the plasma membrane probe are indicated (white arrowheads). Yellow arrows denote mCherry-Mklp1ZEN-4–marked or Myosin IINMY-2–GFP-marked midbody remnants before release from the cell–cell junction. Green arrows mark the same components after release. Schematics illustrate events at each stage. Bars, 5 µm.

Mentions: To monitor contractile ring closure, we collected time-lapse 3D images of embryos expressing a GFP fusion with a pleckstrin homology (PH) domain that binds a phospholipid produced specifically on the plasma membrane (Audhya et al., 2005) and generated end-on views by rotating and projecting the data from the central portion of the embryo (Maddox et al., 2007). The last time when a hole can be detected is ∼280 s after furrow initiation, a point we refer to as apparent closure (Fig. 1 A). To determine whether cytoplasmic isolation is coincident with apparent closure, we loaded caged carboxy-Q-rhodamine–labeled 10-kD dextran into embryos expressing the GFP-tagged plasma membrane probe by injection into the syncytial gonad of adult worms (Fig. S1 B). Diffusion was monitored by imaging at 5-s intervals after photoactivating the probe on one side of the embryo with a pulse of UV light. Before cytokinesis onset, the photoactivated probe equilibrated between the two halves of the embryo with a half-time of ∼40 s; however, after apparent closure, there was no detectable equilibration (Fig. 1 B and Video 1). We conclude that the cytoplasm in the daughter cells becomes diffusionally isolated coincident with the completion of contractile ring constriction.


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)

Abscission occurs in two stages: cytoplasmic isolation and release of the midbody/midbody ring. (A) Furrow diameter was measured in projections of the central region of z stacks of embryos (n = 10) expressing a GFP-tagged plasma membrane probe. (right) Graph plots mean furrow diameter versus time after furrow initiation. Arrow indicates the last time point when a hole can be detected (apparent closure). Error bars are the SDs. (top) Schematics illustrate shape changes during the first division in the C. elegans embryo, highlighting intercellular bridge structure. MTs, microtubules. (B, left) Schematic illustrates the method for monitoring the diffusion of photoactivated dextran between the two half-cells before and after cytokinesis. Examples of probe diffusion in embryos photoactivated before cytokinesis onset (middle; n = 12 embryos) and after apparent closure (right; n = 8 embryos, example shown is 350 s after furrow initiation). Central plane images show the embryos before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). Kymographs were constructed by aligning strips (narrow rectangles) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (C) Central plane fluorescence confocal images of embryos expressing a fluorescently tagged plasma membrane probe (red in merged images) along with either the midbody marker mCherry-Mklp1ZEN-4 (n = 18 embryos) or the midbody ring marker Myosin IINMY-2–GFP (n = 14 embryos). Times are relative to anaphase of the second division (∼900 s after initiation of the first division furrow). Different embryos are shown to illustrate membrane shedding (−80 to 140–s time points) and midbody/midbody ring release (220–260-s time points). White boxes on the low magnification images mark the location of the region shown at higher magnification in the three adjacent panels. Loops and released fragments marked with the plasma membrane probe are indicated (white arrowheads). Yellow arrows denote mCherry-Mklp1ZEN-4–marked or Myosin IINMY-2–GFP-marked midbody remnants before release from the cell–cell junction. Green arrows mark the same components after release. Schematics illustrate events at each stage. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824018&req=5

fig1: Abscission occurs in two stages: cytoplasmic isolation and release of the midbody/midbody ring. (A) Furrow diameter was measured in projections of the central region of z stacks of embryos (n = 10) expressing a GFP-tagged plasma membrane probe. (right) Graph plots mean furrow diameter versus time after furrow initiation. Arrow indicates the last time point when a hole can be detected (apparent closure). Error bars are the SDs. (top) Schematics illustrate shape changes during the first division in the C. elegans embryo, highlighting intercellular bridge structure. MTs, microtubules. (B, left) Schematic illustrates the method for monitoring the diffusion of photoactivated dextran between the two half-cells before and after cytokinesis. Examples of probe diffusion in embryos photoactivated before cytokinesis onset (middle; n = 12 embryos) and after apparent closure (right; n = 8 embryos, example shown is 350 s after furrow initiation). Central plane images show the embryos before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). Kymographs were constructed by aligning strips (narrow rectangles) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (C) Central plane fluorescence confocal images of embryos expressing a fluorescently tagged plasma membrane probe (red in merged images) along with either the midbody marker mCherry-Mklp1ZEN-4 (n = 18 embryos) or the midbody ring marker Myosin IINMY-2–GFP (n = 14 embryos). Times are relative to anaphase of the second division (∼900 s after initiation of the first division furrow). Different embryos are shown to illustrate membrane shedding (−80 to 140–s time points) and midbody/midbody ring release (220–260-s time points). White boxes on the low magnification images mark the location of the region shown at higher magnification in the three adjacent panels. Loops and released fragments marked with the plasma membrane probe are indicated (white arrowheads). Yellow arrows denote mCherry-Mklp1ZEN-4–marked or Myosin IINMY-2–GFP-marked midbody remnants before release from the cell–cell junction. Green arrows mark the same components after release. Schematics illustrate events at each stage. Bars, 5 µm.
Mentions: To monitor contractile ring closure, we collected time-lapse 3D images of embryos expressing a GFP fusion with a pleckstrin homology (PH) domain that binds a phospholipid produced specifically on the plasma membrane (Audhya et al., 2005) and generated end-on views by rotating and projecting the data from the central portion of the embryo (Maddox et al., 2007). The last time when a hole can be detected is ∼280 s after furrow initiation, a point we refer to as apparent closure (Fig. 1 A). To determine whether cytoplasmic isolation is coincident with apparent closure, we loaded caged carboxy-Q-rhodamine–labeled 10-kD dextran into embryos expressing the GFP-tagged plasma membrane probe by injection into the syncytial gonad of adult worms (Fig. S1 B). Diffusion was monitored by imaging at 5-s intervals after photoactivating the probe on one side of the embryo with a pulse of UV light. Before cytokinesis onset, the photoactivated probe equilibrated between the two halves of the embryo with a half-time of ∼40 s; however, after apparent closure, there was no detectable equilibration (Fig. 1 B and Video 1). We conclude that the cytoplasm in the daughter cells becomes diffusionally isolated coincident with the completion of contractile ring constriction.

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