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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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Nascent MRs shed numerous CR components, except F-actin. (A and B) Fixed cells expressing Anillin-GFP and tubulin-mCh stained for anti-Pnut (A) and anti-Rho1 (B) by immunofluorescence. Separated channels of boxed regions are shown magnified on the right. (C) Cell expressing Anillin-GFP and Sticky (Sti)-mCh. (D) Cell expressing Anillin-mCh and Tum-GFP. (E) Cell expressing Anillin-mCh and Pavarotti (Pav)-GFP. (F) Inverted look-up table of the nascent MR of an Aurora B–GFP-expressing cell. (G) The nascent MR of a myosin-GFP–expressing cell. Dashed lines mark the cell boundary. (H) The nascent MR of a cell expressing myosin-GFP (Myo) and Anillin-mCh (Ani). (I) The nascent MR of a cell expressing LifeAct-GFP (LA) and Anillin-mCh. (C, D, E, H, and I) Separated channels of boxed regions are shown magnified at the bottom. (J) The nascent MR of a cell expressing Anillin-GFP fixed and stained with rhodamine-phalloidin. Arrowhead points to an extrusion enriched in Anillin-GFP but lacking actin. Times are hours, minutes, and seconds. Bars: (A [main image] and B–J) 5 µm; (A, magnified images) 1 µm. See also Video 3.
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fig2: Nascent MRs shed numerous CR components, except F-actin. (A and B) Fixed cells expressing Anillin-GFP and tubulin-mCh stained for anti-Pnut (A) and anti-Rho1 (B) by immunofluorescence. Separated channels of boxed regions are shown magnified on the right. (C) Cell expressing Anillin-GFP and Sticky (Sti)-mCh. (D) Cell expressing Anillin-mCh and Tum-GFP. (E) Cell expressing Anillin-mCh and Pavarotti (Pav)-GFP. (F) Inverted look-up table of the nascent MR of an Aurora B–GFP-expressing cell. (G) The nascent MR of a myosin-GFP–expressing cell. Dashed lines mark the cell boundary. (H) The nascent MR of a cell expressing myosin-GFP (Myo) and Anillin-mCh (Ani). (I) The nascent MR of a cell expressing LifeAct-GFP (LA) and Anillin-mCh. (C, D, E, H, and I) Separated channels of boxed regions are shown magnified at the bottom. (J) The nascent MR of a cell expressing Anillin-GFP fixed and stained with rhodamine-phalloidin. Arrowhead points to an extrusion enriched in Anillin-GFP but lacking actin. Times are hours, minutes, and seconds. Bars: (A [main image] and B–J) 5 µm; (A, magnified images) 1 µm. See also Video 3.

Mentions: We wished to determine whether other cytokinesis proteins were also shed with Anillin during MR maturation. Immunofluorescence analysis revealed that the septin, Pnut (Fig. 2 A), and Rho1 (Fig. 2 B) were also enriched on the extruded membranes. Similarly, the Cit-K, Sticky-FP, was extruded with Anillin-FP (Fig. 2 C), as were the components of the centralspindlin complex, RacGAP50C/Tumbleweed (Tum)-FP (Fig. 2 D) and the kinesin-6 motor Pavarotti-FP (Fig. 2 E). Aurora B–GFP, which also localizes primarily to the central spindle and midbody microtubules (Fig. 2 F), was also extruded from the center of the midbody soon after CR closure.


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)

Nascent MRs shed numerous CR components, except F-actin. (A and B) Fixed cells expressing Anillin-GFP and tubulin-mCh stained for anti-Pnut (A) and anti-Rho1 (B) by immunofluorescence. Separated channels of boxed regions are shown magnified on the right. (C) Cell expressing Anillin-GFP and Sticky (Sti)-mCh. (D) Cell expressing Anillin-mCh and Tum-GFP. (E) Cell expressing Anillin-mCh and Pavarotti (Pav)-GFP. (F) Inverted look-up table of the nascent MR of an Aurora B–GFP-expressing cell. (G) The nascent MR of a myosin-GFP–expressing cell. Dashed lines mark the cell boundary. (H) The nascent MR of a cell expressing myosin-GFP (Myo) and Anillin-mCh (Ani). (I) The nascent MR of a cell expressing LifeAct-GFP (LA) and Anillin-mCh. (C, D, E, H, and I) Separated channels of boxed regions are shown magnified at the bottom. (J) The nascent MR of a cell expressing Anillin-GFP fixed and stained with rhodamine-phalloidin. Arrowhead points to an extrusion enriched in Anillin-GFP but lacking actin. Times are hours, minutes, and seconds. Bars: (A [main image] and B–J) 5 µm; (A, magnified images) 1 µm. See also Video 3.
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Related In: Results  -  Collection

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fig2: Nascent MRs shed numerous CR components, except F-actin. (A and B) Fixed cells expressing Anillin-GFP and tubulin-mCh stained for anti-Pnut (A) and anti-Rho1 (B) by immunofluorescence. Separated channels of boxed regions are shown magnified on the right. (C) Cell expressing Anillin-GFP and Sticky (Sti)-mCh. (D) Cell expressing Anillin-mCh and Tum-GFP. (E) Cell expressing Anillin-mCh and Pavarotti (Pav)-GFP. (F) Inverted look-up table of the nascent MR of an Aurora B–GFP-expressing cell. (G) The nascent MR of a myosin-GFP–expressing cell. Dashed lines mark the cell boundary. (H) The nascent MR of a cell expressing myosin-GFP (Myo) and Anillin-mCh (Ani). (I) The nascent MR of a cell expressing LifeAct-GFP (LA) and Anillin-mCh. (C, D, E, H, and I) Separated channels of boxed regions are shown magnified at the bottom. (J) The nascent MR of a cell expressing Anillin-GFP fixed and stained with rhodamine-phalloidin. Arrowhead points to an extrusion enriched in Anillin-GFP but lacking actin. Times are hours, minutes, and seconds. Bars: (A [main image] and B–J) 5 µm; (A, magnified images) 1 µm. See also Video 3.
Mentions: We wished to determine whether other cytokinesis proteins were also shed with Anillin during MR maturation. Immunofluorescence analysis revealed that the septin, Pnut (Fig. 2 A), and Rho1 (Fig. 2 B) were also enriched on the extruded membranes. Similarly, the Cit-K, Sticky-FP, was extruded with Anillin-FP (Fig. 2 C), as were the components of the centralspindlin complex, RacGAP50C/Tumbleweed (Tum)-FP (Fig. 2 D) and the kinesin-6 motor Pavarotti-FP (Fig. 2 E). Aurora B–GFP, which also localizes primarily to the central spindle and midbody microtubules (Fig. 2 F), was also extruded from the center of the midbody soon after CR closure.

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