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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’s essential role during MR formation is as a scaffold. (A) Anti-Sticky (Sti) immunoblot of cell lysates from indicated cell lines after Sticky 3′ UTR dsRNA or LacI control dsRNA incubation, with anti-tubulin blot as a loading control. WT, wild type. (B) Timing of abscission or furrow regression (failure) of cells coexpressing Anillin-GFP and Sticky-mCh or Sticky-KD-mCh, treated for 3–5 d with or without Sticky 3′ UTR dsRNAs. Data are from two independent experiments. (C and D) Time-lapse sequence of a cell coexpressing Anillin-GFP and Sticky-mCh (C) or Sticky-KD-mCh (D). Separated channels of the boxed regions are shown magnified at the bottom. (E) Time-lapse sequence of a cell coexpressing Anillin (Anil)-GFP and Sticky-KD-mCh after depletion of endogenous Sticky. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 9 and 10.
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fig8: Sticky’s essential role during MR formation is as a scaffold. (A) Anti-Sticky (Sti) immunoblot of cell lysates from indicated cell lines after Sticky 3′ UTR dsRNA or LacI control dsRNA incubation, with anti-tubulin blot as a loading control. WT, wild type. (B) Timing of abscission or furrow regression (failure) of cells coexpressing Anillin-GFP and Sticky-mCh or Sticky-KD-mCh, treated for 3–5 d with or without Sticky 3′ UTR dsRNAs. Data are from two independent experiments. (C and D) Time-lapse sequence of a cell coexpressing Anillin-GFP and Sticky-mCh (C) or Sticky-KD-mCh (D). Separated channels of the boxed regions are shown magnified at the bottom. (E) Time-lapse sequence of a cell coexpressing Anillin (Anil)-GFP and Sticky-KD-mCh after depletion of endogenous Sticky. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 9 and 10.

Mentions: We next wished to test whether Sticky kinase activity is required to retain Anillin-GFP at the MR. We generated inducible cell lines that coexpressed Anillin-GFP and mCh-tagged wild-type or kinase-dead (KD) versions of Sticky, at roughly endogenous levels (Fig. 8 A). Both Sticky-mCh and Sticky-KD-mCh were faithfully recruited to the CR and MR, although both also led to the formation of Anillin-GFP–negative, intracellular aggregates, similar to those described by others (Fig. 8, C and D; and Videos 9 and 10; Eda et al., 2001). Expression of either Sticky-mCh or Sticky-KD-mCh rescued the widespread failures of cytokinesis induced by depletion of endogenous Sticky using a dsRNA directed against the Sticky 3′ UTR (Fig. 8 B). That formation of Sticky- and Anillin-positive MRs (Fig. 8, C–E; and Video 10) were rescued in Sticky-KD-mCh cells indicates that Sticky kinase activity is dispensable for both MR formation and abscission (Fig. 8, B and E). However, abscission timing was slightly accelerated for Sticky-KD-mCh MRs (128 ± 49 min, mean ± SD; n = 28) than for Sticky-mCh MRs (196 ± 67 min, mean ± SD; n = 26). Furthermore, the majority of cells that failed did not recruit detectable levels of Sticky-mCh (7/8 failures) or Sticky-KD-mCh (18/24 failures) to the CR/MR, consistent with failures reflecting the lack of Sticky rather than the lack of kinase function (Fig. 8 B and Fig. S3 B). In addition, excess Sticky-mCh and Sticky-KD-mCh were both also internalized and extruded/shed with Anillin-GFP (Fig. 8, C–E; and Videos 9 and 10), indicating that Sticky must itself be subject to retention at the midbody. We conclude that the essential role of Sticky in these cells is as a scaffold that serves to retain Anillin and stabilize the 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’s essential role during MR formation is as a scaffold. (A) Anti-Sticky (Sti) immunoblot of cell lysates from indicated cell lines after Sticky 3′ UTR dsRNA or LacI control dsRNA incubation, with anti-tubulin blot as a loading control. WT, wild type. (B) Timing of abscission or furrow regression (failure) of cells coexpressing Anillin-GFP and Sticky-mCh or Sticky-KD-mCh, treated for 3–5 d with or without Sticky 3′ UTR dsRNAs. Data are from two independent experiments. (C and D) Time-lapse sequence of a cell coexpressing Anillin-GFP and Sticky-mCh (C) or Sticky-KD-mCh (D). Separated channels of the boxed regions are shown magnified at the bottom. (E) Time-lapse sequence of a cell coexpressing Anillin (Anil)-GFP and Sticky-KD-mCh after depletion of endogenous Sticky. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 9 and 10.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: Sticky’s essential role during MR formation is as a scaffold. (A) Anti-Sticky (Sti) immunoblot of cell lysates from indicated cell lines after Sticky 3′ UTR dsRNA or LacI control dsRNA incubation, with anti-tubulin blot as a loading control. WT, wild type. (B) Timing of abscission or furrow regression (failure) of cells coexpressing Anillin-GFP and Sticky-mCh or Sticky-KD-mCh, treated for 3–5 d with or without Sticky 3′ UTR dsRNAs. Data are from two independent experiments. (C and D) Time-lapse sequence of a cell coexpressing Anillin-GFP and Sticky-mCh (C) or Sticky-KD-mCh (D). Separated channels of the boxed regions are shown magnified at the bottom. (E) Time-lapse sequence of a cell coexpressing Anillin (Anil)-GFP and Sticky-KD-mCh after depletion of endogenous Sticky. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 9 and 10.
Mentions: We next wished to test whether Sticky kinase activity is required to retain Anillin-GFP at the MR. We generated inducible cell lines that coexpressed Anillin-GFP and mCh-tagged wild-type or kinase-dead (KD) versions of Sticky, at roughly endogenous levels (Fig. 8 A). Both Sticky-mCh and Sticky-KD-mCh were faithfully recruited to the CR and MR, although both also led to the formation of Anillin-GFP–negative, intracellular aggregates, similar to those described by others (Fig. 8, C and D; and Videos 9 and 10; Eda et al., 2001). Expression of either Sticky-mCh or Sticky-KD-mCh rescued the widespread failures of cytokinesis induced by depletion of endogenous Sticky using a dsRNA directed against the Sticky 3′ UTR (Fig. 8 B). That formation of Sticky- and Anillin-positive MRs (Fig. 8, C–E; and Video 10) were rescued in Sticky-KD-mCh cells indicates that Sticky kinase activity is dispensable for both MR formation and abscission (Fig. 8, B and E). However, abscission timing was slightly accelerated for Sticky-KD-mCh MRs (128 ± 49 min, mean ± SD; n = 28) than for Sticky-mCh MRs (196 ± 67 min, mean ± SD; n = 26). Furthermore, the majority of cells that failed did not recruit detectable levels of Sticky-mCh (7/8 failures) or Sticky-KD-mCh (18/24 failures) to the CR/MR, consistent with failures reflecting the lack of Sticky rather than the lack of kinase function (Fig. 8 B and Fig. S3 B). In addition, excess Sticky-mCh and Sticky-KD-mCh were both also internalized and extruded/shed with Anillin-GFP (Fig. 8, C–E; and Videos 9 and 10), indicating that Sticky must itself be subject to retention at the midbody. We conclude that the essential role of Sticky in these cells is as a scaffold that serves to retain Anillin and stabilize the 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