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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 to limit extrusion/shedding and retain Anillin at the MR. (A and B) Sticky-depleted cells expressing Anillin-GFP and mCh-tubulin failing cytokinesis (A) or prematurely abscising (B) (63× objective). Boxed regions are shown magnified and with separated channels below. (C) Quantification of relative Anillin-GFP intensities (sum intensity) at the MR from the time of midbody formation in control (n = 9) and Sticky-depleted cells (n = 17). (D) Quantification of individual extrusion or internalization events (open circles) plotted as a function of time and associated Anillin-GFP log10 intensity (as a measure of total intensity at the close of furrowing, t = 0). Closed circles represent mean values for each 10-min interval (n = 9 cells each, from one experiment representative of two repeats). (E) Timing of abscission or furrow regression (failure) events of individual Anillin-GFP cells treated for 30–48 h with or without Sticky dsRNAs. Data are from two independent experiments, horizontal lines mark the mean times, and error bars show the SDs. (F–H) Confocal images of Sticky-depleted cells expressing Anillin-GFP (left), stained for Pnut (F, center), Rho1 (G, left), or F-actin (H, center) and DNA. (I and J) Confocal images of untransfected S2 cells treated with LacI control dsRNA (I) or Sticky dsRNA (J) and stained for endogenous Anillin (left), tubulin (center), and DNA (63× objective). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 6 and 7.
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fig6: Sticky acts to limit extrusion/shedding and retain Anillin at the MR. (A and B) Sticky-depleted cells expressing Anillin-GFP and mCh-tubulin failing cytokinesis (A) or prematurely abscising (B) (63× objective). Boxed regions are shown magnified and with separated channels below. (C) Quantification of relative Anillin-GFP intensities (sum intensity) at the MR from the time of midbody formation in control (n = 9) and Sticky-depleted cells (n = 17). (D) Quantification of individual extrusion or internalization events (open circles) plotted as a function of time and associated Anillin-GFP log10 intensity (as a measure of total intensity at the close of furrowing, t = 0). Closed circles represent mean values for each 10-min interval (n = 9 cells each, from one experiment representative of two repeats). (E) Timing of abscission or furrow regression (failure) events of individual Anillin-GFP cells treated for 30–48 h with or without Sticky dsRNAs. Data are from two independent experiments, horizontal lines mark the mean times, and error bars show the SDs. (F–H) Confocal images of Sticky-depleted cells expressing Anillin-GFP (left), stained for Pnut (F, center), Rho1 (G, left), or F-actin (H, center) and DNA. (I and J) Confocal images of untransfected S2 cells treated with LacI control dsRNA (I) or Sticky dsRNA (J) and stained for endogenous Anillin (left), tubulin (center), and DNA (63× objective). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 6 and 7.

Mentions: Time-lapse sequences captured at 1-min intervals with a 63× objective revealed that Sticky-depleted CRs closed with normal kinetics and morphology (Fig. 6, A and B; and Videos 6 and 7), consistent with previous studies (Echard et al., 2004; Bassi et al., 2011). The midbody microtubules also adopted their characteristic compacted morphology at the close of furrowing (Fig. 6, A and B), unlike Anillin-depleted cells (Fig. 3, B and D; Kechad et al., 2012). However, measuring Anillin-GFP intensities revealed that Anillin levels continued to increase after the close of furrowing, such that the peak intensity of the nascent MR was ∼10 min later than that of control cells (Fig. 6 C). At the time of normal MR formation and maturation, Anillin-GFP displayed enhanced extrusion and internalization events (Fig. 6 D). As in controls, blebs and tubules of Anillin-positive membranes formed and subsequently left the MR region by lateral movement or outright shedding (Fig. 6, A and B; and Videos 6 and 7). Although we observed clear examples of both internalization and extrusion/shedding in both control and Sticky-depleted cells, it was often difficult to differentiate between the two in any given case, especially as extruded material could subsequently be internalized. We therefore simply quantified removal (by either mechanism). This revealed that more Anillin-GFP was removed from Sticky-depleted nascent MRs and over a longer period than in controls (Fig. 6 D). Removal continued until Anillin-GFP was no longer detectable at the MR site, and furrows either regressed within a few minutes or underwent abscission prematurely (Fig. 6, A and B; and Videos 6 and 7).


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 to limit extrusion/shedding and retain Anillin at the MR. (A and B) Sticky-depleted cells expressing Anillin-GFP and mCh-tubulin failing cytokinesis (A) or prematurely abscising (B) (63× objective). Boxed regions are shown magnified and with separated channels below. (C) Quantification of relative Anillin-GFP intensities (sum intensity) at the MR from the time of midbody formation in control (n = 9) and Sticky-depleted cells (n = 17). (D) Quantification of individual extrusion or internalization events (open circles) plotted as a function of time and associated Anillin-GFP log10 intensity (as a measure of total intensity at the close of furrowing, t = 0). Closed circles represent mean values for each 10-min interval (n = 9 cells each, from one experiment representative of two repeats). (E) Timing of abscission or furrow regression (failure) events of individual Anillin-GFP cells treated for 30–48 h with or without Sticky dsRNAs. Data are from two independent experiments, horizontal lines mark the mean times, and error bars show the SDs. (F–H) Confocal images of Sticky-depleted cells expressing Anillin-GFP (left), stained for Pnut (F, center), Rho1 (G, left), or F-actin (H, center) and DNA. (I and J) Confocal images of untransfected S2 cells treated with LacI control dsRNA (I) or Sticky dsRNA (J) and stained for endogenous Anillin (left), tubulin (center), and DNA (63× objective). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 6 and 7.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Sticky acts to limit extrusion/shedding and retain Anillin at the MR. (A and B) Sticky-depleted cells expressing Anillin-GFP and mCh-tubulin failing cytokinesis (A) or prematurely abscising (B) (63× objective). Boxed regions are shown magnified and with separated channels below. (C) Quantification of relative Anillin-GFP intensities (sum intensity) at the MR from the time of midbody formation in control (n = 9) and Sticky-depleted cells (n = 17). (D) Quantification of individual extrusion or internalization events (open circles) plotted as a function of time and associated Anillin-GFP log10 intensity (as a measure of total intensity at the close of furrowing, t = 0). Closed circles represent mean values for each 10-min interval (n = 9 cells each, from one experiment representative of two repeats). (E) Timing of abscission or furrow regression (failure) events of individual Anillin-GFP cells treated for 30–48 h with or without Sticky dsRNAs. Data are from two independent experiments, horizontal lines mark the mean times, and error bars show the SDs. (F–H) Confocal images of Sticky-depleted cells expressing Anillin-GFP (left), stained for Pnut (F, center), Rho1 (G, left), or F-actin (H, center) and DNA. (I and J) Confocal images of untransfected S2 cells treated with LacI control dsRNA (I) or Sticky dsRNA (J) and stained for endogenous Anillin (left), tubulin (center), and DNA (63× objective). Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Videos 6 and 7.
Mentions: Time-lapse sequences captured at 1-min intervals with a 63× objective revealed that Sticky-depleted CRs closed with normal kinetics and morphology (Fig. 6, A and B; and Videos 6 and 7), consistent with previous studies (Echard et al., 2004; Bassi et al., 2011). The midbody microtubules also adopted their characteristic compacted morphology at the close of furrowing (Fig. 6, A and B), unlike Anillin-depleted cells (Fig. 3, B and D; Kechad et al., 2012). However, measuring Anillin-GFP intensities revealed that Anillin levels continued to increase after the close of furrowing, such that the peak intensity of the nascent MR was ∼10 min later than that of control cells (Fig. 6 C). At the time of normal MR formation and maturation, Anillin-GFP displayed enhanced extrusion and internalization events (Fig. 6 D). As in controls, blebs and tubules of Anillin-positive membranes formed and subsequently left the MR region by lateral movement or outright shedding (Fig. 6, A and B; and Videos 6 and 7). Although we observed clear examples of both internalization and extrusion/shedding in both control and Sticky-depleted cells, it was often difficult to differentiate between the two in any given case, especially as extruded material could subsequently be internalized. We therefore simply quantified removal (by either mechanism). This revealed that more Anillin-GFP was removed from Sticky-depleted nascent MRs and over a longer period than in controls (Fig. 6 D). Removal continued until Anillin-GFP was no longer detectable at the MR site, and furrows either regressed within a few minutes or underwent abscission prematurely (Fig. 6, A and B; and Videos 6 and 7).

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