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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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Proper maturation of the MR requires septin-dependent removal of Anillin via its C-terminal PHD. (A) Frequency of extrusion from time-lapse sequences of Anillin-ΔPH-GFP and of Anillin-GFP after 8-d Pnut RNAi. (B) Anti-Pnut immunoblot of S2 cell lysates serially diluted after LacI control dsRNA incubation or after Pnut dsRNA incubation; anti-tubulin is the loading control. (C) Time-lapse sequences of cells expressing Anillin-ΔPH-GFP depleted of endogenous Anillin. (D) Time-lapse sequence of an Anillin-GFP–expressing cell after 8-d Pnut RNAi. (E) Representative images of age-matched MR structures from Anillin-GFP or Anillin-ΔPH-GFP cells treated with the indicated dsRNAs. (F) Volume measurements of the nascent MR of cells expressing Anillin-ΔPH-GFP, Anillin-ΔC-GFP, or Anillin-GFP treated with the indicated dsRNAs, from the end of furrowing, normalized at each time point to equivalently aged Anillin-GFP controls (n = 10 per condition from two independent experiments). (G) Timing of abscission (abscis.) or furrow regression (fail) of Anillin-GFP cells treated for 7–9 d with LacI (control, n = 30) or Pnut dsRNAs (n = 75) and of Anillin-ΔPH-GFP cells treated for 3 d with LacI (control, n = 30) or Anillin dsRNAs (n = 75). Mean values are shown; data are from a single representative experiment out of three repeats. Times are given in hours, minutes, and seconds. Bars: (C and D) 5 µm; (E) 1 µm.
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fig5: Proper maturation of the MR requires septin-dependent removal of Anillin via its C-terminal PHD. (A) Frequency of extrusion from time-lapse sequences of Anillin-ΔPH-GFP and of Anillin-GFP after 8-d Pnut RNAi. (B) Anti-Pnut immunoblot of S2 cell lysates serially diluted after LacI control dsRNA incubation or after Pnut dsRNA incubation; anti-tubulin is the loading control. (C) Time-lapse sequences of cells expressing Anillin-ΔPH-GFP depleted of endogenous Anillin. (D) Time-lapse sequence of an Anillin-GFP–expressing cell after 8-d Pnut RNAi. (E) Representative images of age-matched MR structures from Anillin-GFP or Anillin-ΔPH-GFP cells treated with the indicated dsRNAs. (F) Volume measurements of the nascent MR of cells expressing Anillin-ΔPH-GFP, Anillin-ΔC-GFP, or Anillin-GFP treated with the indicated dsRNAs, from the end of furrowing, normalized at each time point to equivalently aged Anillin-GFP controls (n = 10 per condition from two independent experiments). (G) Timing of abscission (abscis.) or furrow regression (fail) of Anillin-GFP cells treated for 7–9 d with LacI (control, n = 30) or Pnut dsRNAs (n = 75) and of Anillin-ΔPH-GFP cells treated for 3 d with LacI (control, n = 30) or Anillin dsRNAs (n = 75). Mean values are shown; data are from a single representative experiment out of three repeats. Times are given in hours, minutes, and seconds. Bars: (C and D) 5 µm; (E) 1 µm.

Mentions: The Anillin PHD is known to bind septins (Field and Alberts, 1995; Oegema et al., 2000; Liu et al., 2012) and phosphoinositides (Liu et al., 2012), and septins are required for the recruitment of Anillin-ΔN to the cleavage furrow (Kechad et al., 2012). We further evaluated the requirement for the PHD and septins in Anillin removal during MR maturation. Anillin-ΔPH-FP localized efficiently to the CR and MR but showed little, if any, evidence of extrusion or internalization from the nascent MR, whether or not endogenous Anillin was depleted (Fig. 5, A and C). Anillin-ΔPH-GFP MRs also appeared larger than controls of the same age (Fig. 5 E). Measuring MR volumes over time revealed that Anillin-ΔPH-GFP MRs did not thin to the same extent as Anillin-GFP controls (Fig. 5 F). This effect was dominant, suggesting that the PHD is autonomously required for Anillin removal. A 7–8-d incubation with double-stranded RNAs (dsRNAs) targeting the septin Pnut (Neufeld and Rubin, 1994), which results in >94% depletion in S2 cells (Fig. 5 B), also blocked extrusion/ shedding of Anillin-GFP (Fig. 5 A) and inhibited the thinning of the MR during its maturation (Fig. 5, D–F). Thus, the PHD of Anillin and Pnut are each required for cortical removal during MR maturation.


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)

Proper maturation of the MR requires septin-dependent removal of Anillin via its C-terminal PHD. (A) Frequency of extrusion from time-lapse sequences of Anillin-ΔPH-GFP and of Anillin-GFP after 8-d Pnut RNAi. (B) Anti-Pnut immunoblot of S2 cell lysates serially diluted after LacI control dsRNA incubation or after Pnut dsRNA incubation; anti-tubulin is the loading control. (C) Time-lapse sequences of cells expressing Anillin-ΔPH-GFP depleted of endogenous Anillin. (D) Time-lapse sequence of an Anillin-GFP–expressing cell after 8-d Pnut RNAi. (E) Representative images of age-matched MR structures from Anillin-GFP or Anillin-ΔPH-GFP cells treated with the indicated dsRNAs. (F) Volume measurements of the nascent MR of cells expressing Anillin-ΔPH-GFP, Anillin-ΔC-GFP, or Anillin-GFP treated with the indicated dsRNAs, from the end of furrowing, normalized at each time point to equivalently aged Anillin-GFP controls (n = 10 per condition from two independent experiments). (G) Timing of abscission (abscis.) or furrow regression (fail) of Anillin-GFP cells treated for 7–9 d with LacI (control, n = 30) or Pnut dsRNAs (n = 75) and of Anillin-ΔPH-GFP cells treated for 3 d with LacI (control, n = 30) or Anillin dsRNAs (n = 75). Mean values are shown; data are from a single representative experiment out of three repeats. Times are given in hours, minutes, and seconds. Bars: (C and D) 5 µm; (E) 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Proper maturation of the MR requires septin-dependent removal of Anillin via its C-terminal PHD. (A) Frequency of extrusion from time-lapse sequences of Anillin-ΔPH-GFP and of Anillin-GFP after 8-d Pnut RNAi. (B) Anti-Pnut immunoblot of S2 cell lysates serially diluted after LacI control dsRNA incubation or after Pnut dsRNA incubation; anti-tubulin is the loading control. (C) Time-lapse sequences of cells expressing Anillin-ΔPH-GFP depleted of endogenous Anillin. (D) Time-lapse sequence of an Anillin-GFP–expressing cell after 8-d Pnut RNAi. (E) Representative images of age-matched MR structures from Anillin-GFP or Anillin-ΔPH-GFP cells treated with the indicated dsRNAs. (F) Volume measurements of the nascent MR of cells expressing Anillin-ΔPH-GFP, Anillin-ΔC-GFP, or Anillin-GFP treated with the indicated dsRNAs, from the end of furrowing, normalized at each time point to equivalently aged Anillin-GFP controls (n = 10 per condition from two independent experiments). (G) Timing of abscission (abscis.) or furrow regression (fail) of Anillin-GFP cells treated for 7–9 d with LacI (control, n = 30) or Pnut dsRNAs (n = 75) and of Anillin-ΔPH-GFP cells treated for 3 d with LacI (control, n = 30) or Anillin dsRNAs (n = 75). Mean values are shown; data are from a single representative experiment out of three repeats. Times are given in hours, minutes, and seconds. Bars: (C and D) 5 µm; (E) 1 µm.
Mentions: The Anillin PHD is known to bind septins (Field and Alberts, 1995; Oegema et al., 2000; Liu et al., 2012) and phosphoinositides (Liu et al., 2012), and septins are required for the recruitment of Anillin-ΔN to the cleavage furrow (Kechad et al., 2012). We further evaluated the requirement for the PHD and septins in Anillin removal during MR maturation. Anillin-ΔPH-FP localized efficiently to the CR and MR but showed little, if any, evidence of extrusion or internalization from the nascent MR, whether or not endogenous Anillin was depleted (Fig. 5, A and C). Anillin-ΔPH-GFP MRs also appeared larger than controls of the same age (Fig. 5 E). Measuring MR volumes over time revealed that Anillin-ΔPH-GFP MRs did not thin to the same extent as Anillin-GFP controls (Fig. 5 F). This effect was dominant, suggesting that the PHD is autonomously required for Anillin removal. A 7–8-d incubation with double-stranded RNAs (dsRNAs) targeting the septin Pnut (Neufeld and Rubin, 1994), which results in >94% depletion in S2 cells (Fig. 5 B), also blocked extrusion/ shedding of Anillin-GFP (Fig. 5 A) and inhibited the thinning of the MR during its maturation (Fig. 5, D–F). Thus, the PHD of Anillin and Pnut are each required for cortical removal during MR maturation.

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