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Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring.

El Amine N, Kechad A, Jananji S, Hickson GR - J. Cell Biol. (2013)

Bottom Line: During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms.The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR.Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Cancérologie Charles Bruneau, Centre Hospitalier Universitaire Sainte-Justine Centre de Recherche, Montréal, Québec H3T 1C5, Canada.

ABSTRACT
During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms. Using time-lapse microscopy of Drosophila melanogaster S2 cells, we show that the transition from the CR to the MR proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of the scaffold protein Anillin. The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR. Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process. Our findings highlight the relatedness of the CR and MR and suggest that membrane removal is coordinated with CR disassembly.

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Sticky acts with the N terminus of Anillin to promote both MR formation and CR stability. (A and B) Time-lapse sequence of cells expressing mCh-tubulin and Anillin-ΔN-GFP (A) or Anillin-ΔC-GFP (B) after a ∼30-h incubation with Sticky dsRNAs (63× objective and 2 × 2 camera binning). (C) Quantification from time-lapse records of failed attempts at cytokinesis that form internal Anillin-ΔC-FP–positive MR-like structures after incubation with the indicated dsRNAs. Data are from two independent experiments. (D and E) Confocal images of cells coexpressing Sticky-mCh and Anillin-ΔC-GFP (D) or Anillin-ΔN-GFP (E) after endogenous Anillin depletion. Dotted line in D marks the cell boundary (63× objective and no camera binning). (F) Time-lapse sequence of a cell expressing Anillin-ΔC after codepletion of Sticky and Anillin. (G) Time-lapse sequence of a cell expressing Anillin-GFP after codepletion of Pnut (144–162-h RNAi) and Sticky (30–48-h RNAi), captured with a 63× objective with 2 × 2 camera binning. Closed arrowheads mark a presumed MR remnant from a previous division. Open arrowheads mark failing MR from the current division attempt. (H) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of the percentage of Anillin-GFP and Anillin-ΔC-GFP cells displaying oscillating furrows during CR closure after incubation with the indicated dsRNAs (n > 78 per condition from two independent experiments). (I) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of attempts at cytokinesis that result in Anillin-GFP–positive MR structures after incubation with indicated dsRNAs (n > 45 per condition, from two independent experiments). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 8.
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fig7: Sticky acts with the N terminus of Anillin to promote both MR formation and CR stability. (A and B) Time-lapse sequence of cells expressing mCh-tubulin and Anillin-ΔN-GFP (A) or Anillin-ΔC-GFP (B) after a ∼30-h incubation with Sticky dsRNAs (63× objective and 2 × 2 camera binning). (C) Quantification from time-lapse records of failed attempts at cytokinesis that form internal Anillin-ΔC-FP–positive MR-like structures after incubation with the indicated dsRNAs. Data are from two independent experiments. (D and E) Confocal images of cells coexpressing Sticky-mCh and Anillin-ΔC-GFP (D) or Anillin-ΔN-GFP (E) after endogenous Anillin depletion. Dotted line in D marks the cell boundary (63× objective and no camera binning). (F) Time-lapse sequence of a cell expressing Anillin-ΔC after codepletion of Sticky and Anillin. (G) Time-lapse sequence of a cell expressing Anillin-GFP after codepletion of Pnut (144–162-h RNAi) and Sticky (30–48-h RNAi), captured with a 63× objective with 2 × 2 camera binning. Closed arrowheads mark a presumed MR remnant from a previous division. Open arrowheads mark failing MR from the current division attempt. (H) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of the percentage of Anillin-GFP and Anillin-ΔC-GFP cells displaying oscillating furrows during CR closure after incubation with the indicated dsRNAs (n > 78 per condition from two independent experiments). (I) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of attempts at cytokinesis that result in Anillin-GFP–positive MR structures after incubation with indicated dsRNAs (n > 45 per condition, from two independent experiments). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 8.

Mentions: Sticky depletion had no effect on the recruitment or removal of Anillin-ΔN-FP (Fig. 7 A) but greatly diminished the localization of Anillin-ΔC-FP to the nascent MR (Fig. 7 B) and prevented it from forming stable MR-like structures, whether or not endogenous Anillin was codepleted (Fig. 7 C). Furthermore, a functional Sticky-mCh localized to internal Anillin-ΔC-GFP–dependent MR-like structures that resulted upon depletion of endogenous Anillin (Fig. 7 D) but did not colocalize with Anillin-ΔN-GFP–positive structures that accumulate around the nascent MR when endogenous Anillin is present (Fig. 7 E). Thus, Sticky is specifically required to retain the N terminus of Anillin at the mature MR.


Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring.

El Amine N, Kechad A, Jananji S, Hickson GR - J. Cell Biol. (2013)

Sticky acts with the N terminus of Anillin to promote both MR formation and CR stability. (A and B) Time-lapse sequence of cells expressing mCh-tubulin and Anillin-ΔN-GFP (A) or Anillin-ΔC-GFP (B) after a ∼30-h incubation with Sticky dsRNAs (63× objective and 2 × 2 camera binning). (C) Quantification from time-lapse records of failed attempts at cytokinesis that form internal Anillin-ΔC-FP–positive MR-like structures after incubation with the indicated dsRNAs. Data are from two independent experiments. (D and E) Confocal images of cells coexpressing Sticky-mCh and Anillin-ΔC-GFP (D) or Anillin-ΔN-GFP (E) after endogenous Anillin depletion. Dotted line in D marks the cell boundary (63× objective and no camera binning). (F) Time-lapse sequence of a cell expressing Anillin-ΔC after codepletion of Sticky and Anillin. (G) Time-lapse sequence of a cell expressing Anillin-GFP after codepletion of Pnut (144–162-h RNAi) and Sticky (30–48-h RNAi), captured with a 63× objective with 2 × 2 camera binning. Closed arrowheads mark a presumed MR remnant from a previous division. Open arrowheads mark failing MR from the current division attempt. (H) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of the percentage of Anillin-GFP and Anillin-ΔC-GFP cells displaying oscillating furrows during CR closure after incubation with the indicated dsRNAs (n > 78 per condition from two independent experiments). (I) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of attempts at cytokinesis that result in Anillin-GFP–positive MR structures after incubation with indicated dsRNAs (n > 45 per condition, from two independent experiments). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 8.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824009&req=5

fig7: Sticky acts with the N terminus of Anillin to promote both MR formation and CR stability. (A and B) Time-lapse sequence of cells expressing mCh-tubulin and Anillin-ΔN-GFP (A) or Anillin-ΔC-GFP (B) after a ∼30-h incubation with Sticky dsRNAs (63× objective and 2 × 2 camera binning). (C) Quantification from time-lapse records of failed attempts at cytokinesis that form internal Anillin-ΔC-FP–positive MR-like structures after incubation with the indicated dsRNAs. Data are from two independent experiments. (D and E) Confocal images of cells coexpressing Sticky-mCh and Anillin-ΔC-GFP (D) or Anillin-ΔN-GFP (E) after endogenous Anillin depletion. Dotted line in D marks the cell boundary (63× objective and no camera binning). (F) Time-lapse sequence of a cell expressing Anillin-ΔC after codepletion of Sticky and Anillin. (G) Time-lapse sequence of a cell expressing Anillin-GFP after codepletion of Pnut (144–162-h RNAi) and Sticky (30–48-h RNAi), captured with a 63× objective with 2 × 2 camera binning. Closed arrowheads mark a presumed MR remnant from a previous division. Open arrowheads mark failing MR from the current division attempt. (H) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of the percentage of Anillin-GFP and Anillin-ΔC-GFP cells displaying oscillating furrows during CR closure after incubation with the indicated dsRNAs (n > 78 per condition from two independent experiments). (I) Quantification from time-lapse records (40× objective and 2 × 2 camera binning) of attempts at cytokinesis that result in Anillin-GFP–positive MR structures after incubation with indicated dsRNAs (n > 45 per condition, from two independent experiments). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 8.
Mentions: Sticky depletion had no effect on the recruitment or removal of Anillin-ΔN-FP (Fig. 7 A) but greatly diminished the localization of Anillin-ΔC-FP to the nascent MR (Fig. 7 B) and prevented it from forming stable MR-like structures, whether or not endogenous Anillin was codepleted (Fig. 7 C). Furthermore, a functional Sticky-mCh localized to internal Anillin-ΔC-GFP–dependent MR-like structures that resulted upon depletion of endogenous Anillin (Fig. 7 D) but did not colocalize with Anillin-ΔN-GFP–positive structures that accumulate around the nascent MR when endogenous Anillin is present (Fig. 7 E). Thus, Sticky is specifically required to retain the N terminus of Anillin at the mature MR.

Bottom Line: During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms.The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR.Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Cancérologie Charles Bruneau, Centre Hospitalier Universitaire Sainte-Justine Centre de Recherche, Montréal, Québec H3T 1C5, Canada.

ABSTRACT
During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms. Using time-lapse microscopy of Drosophila melanogaster S2 cells, we show that the transition from the CR to the MR proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of the scaffold protein Anillin. The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR. Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process. Our findings highlight the relatedness of the CR and MR and suggest that membrane removal is coordinated with CR disassembly.

Show MeSH
Related in: MedlinePlus