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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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Shedding from the nascent MR requires Anillin but not ESCRT-III. (A and B) Time-lapse sequence of cells expressing Aurora B–GFP and mCh-tubulin after a 3-d incubation without (A) or with (B) Anillin dsRNAs, acquired with a 100×, 1.4 NA objective. Arrowheads in A mark shedding events. (C and D) Time-lapse sequence of cells expressing Tum-GFP and mCh-tubulin after a 3-d incubation without (C) or with (D) Anillin dsRNAs. Arrowheads in C mark shedding events. (E and F) Selected frames from a time-lapse sequence of Anillin-GFP cells after a 4-d incubation without (E) or with (F) shrub dsRNAs, acquired with a 40× objective. Dotted circles highlight sister cells; yellow arrowheads mark mature MRs/MR remnants from previous divisions; and white arrowheads mark MRs from the current division. (G) Quantification of Anillin-GFP–expressing cells that have failed to undergo abscission and thus remain paired at metaphase in control and Shrub-depleted cells. Data are from two independent experiments. (H) Selected frames from a time-lapse sequence of Anillin-GFP cells, acquired with a 100× objective, after a 4-d incubation with shrub dsRNAs. Boxed regions are shown magnified and with separated channels on the right; yellow arrowheads mark mature MRs from previous divisions; and white arrowheads mark nascent MRs from the current division that are shedding Anillin-GFP. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 4.
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fig3: Shedding from the nascent MR requires Anillin but not ESCRT-III. (A and B) Time-lapse sequence of cells expressing Aurora B–GFP and mCh-tubulin after a 3-d incubation without (A) or with (B) Anillin dsRNAs, acquired with a 100×, 1.4 NA objective. Arrowheads in A mark shedding events. (C and D) Time-lapse sequence of cells expressing Tum-GFP and mCh-tubulin after a 3-d incubation without (C) or with (D) Anillin dsRNAs. Arrowheads in C mark shedding events. (E and F) Selected frames from a time-lapse sequence of Anillin-GFP cells after a 4-d incubation without (E) or with (F) shrub dsRNAs, acquired with a 40× objective. Dotted circles highlight sister cells; yellow arrowheads mark mature MRs/MR remnants from previous divisions; and white arrowheads mark MRs from the current division. (G) Quantification of Anillin-GFP–expressing cells that have failed to undergo abscission and thus remain paired at metaphase in control and Shrub-depleted cells. Data are from two independent experiments. (H) Selected frames from a time-lapse sequence of Anillin-GFP cells, acquired with a 100× objective, after a 4-d incubation with shrub dsRNAs. Boxed regions are shown magnified and with separated channels on the right; yellow arrowheads mark mature MRs from previous divisions; and white arrowheads mark nascent MRs from the current division that are shedding Anillin-GFP. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 4.

Mentions: Aurora B–GFP and Tum-GFP showed evidence of shedding even when Anillin was not overexpressed (Fig. 3, A and C). We therefore used these markers to test whether Anillin was itself required for shedding. In Anillin-depleted cells, Aurora B–GFP was no longer extruded compared with controls but still disappeared from the cleavage site over a similar time course to controls (n > 20; Fig. 3, A and B). Similarly, no evidence of Tum-GFP extrusion was observed from Anillin-depleted late CRs/nascent MRs (n > 20; Fig. 3, C and D). Thus, Anillin is required for extrusion, although we note that this may reflect a direct requirement for Anillin in the extrusion process, an indirect consequence of the requirement for Anillin in complete CR closure (Kechad et al., 2012), or both.


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)

Shedding from the nascent MR requires Anillin but not ESCRT-III. (A and B) Time-lapse sequence of cells expressing Aurora B–GFP and mCh-tubulin after a 3-d incubation without (A) or with (B) Anillin dsRNAs, acquired with a 100×, 1.4 NA objective. Arrowheads in A mark shedding events. (C and D) Time-lapse sequence of cells expressing Tum-GFP and mCh-tubulin after a 3-d incubation without (C) or with (D) Anillin dsRNAs. Arrowheads in C mark shedding events. (E and F) Selected frames from a time-lapse sequence of Anillin-GFP cells after a 4-d incubation without (E) or with (F) shrub dsRNAs, acquired with a 40× objective. Dotted circles highlight sister cells; yellow arrowheads mark mature MRs/MR remnants from previous divisions; and white arrowheads mark MRs from the current division. (G) Quantification of Anillin-GFP–expressing cells that have failed to undergo abscission and thus remain paired at metaphase in control and Shrub-depleted cells. Data are from two independent experiments. (H) Selected frames from a time-lapse sequence of Anillin-GFP cells, acquired with a 100× objective, after a 4-d incubation with shrub dsRNAs. Boxed regions are shown magnified and with separated channels on the right; yellow arrowheads mark mature MRs from previous divisions; and white arrowheads mark nascent MRs from the current division that are shedding Anillin-GFP. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 4.
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Related In: Results  -  Collection

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fig3: Shedding from the nascent MR requires Anillin but not ESCRT-III. (A and B) Time-lapse sequence of cells expressing Aurora B–GFP and mCh-tubulin after a 3-d incubation without (A) or with (B) Anillin dsRNAs, acquired with a 100×, 1.4 NA objective. Arrowheads in A mark shedding events. (C and D) Time-lapse sequence of cells expressing Tum-GFP and mCh-tubulin after a 3-d incubation without (C) or with (D) Anillin dsRNAs. Arrowheads in C mark shedding events. (E and F) Selected frames from a time-lapse sequence of Anillin-GFP cells after a 4-d incubation without (E) or with (F) shrub dsRNAs, acquired with a 40× objective. Dotted circles highlight sister cells; yellow arrowheads mark mature MRs/MR remnants from previous divisions; and white arrowheads mark MRs from the current division. (G) Quantification of Anillin-GFP–expressing cells that have failed to undergo abscission and thus remain paired at metaphase in control and Shrub-depleted cells. Data are from two independent experiments. (H) Selected frames from a time-lapse sequence of Anillin-GFP cells, acquired with a 100× objective, after a 4-d incubation with shrub dsRNAs. Boxed regions are shown magnified and with separated channels on the right; yellow arrowheads mark mature MRs from previous divisions; and white arrowheads mark nascent MRs from the current division that are shedding Anillin-GFP. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 4.
Mentions: Aurora B–GFP and Tum-GFP showed evidence of shedding even when Anillin was not overexpressed (Fig. 3, A and C). We therefore used these markers to test whether Anillin was itself required for shedding. In Anillin-depleted cells, Aurora B–GFP was no longer extruded compared with controls but still disappeared from the cleavage site over a similar time course to controls (n > 20; Fig. 3, A and B). Similarly, no evidence of Tum-GFP extrusion was observed from Anillin-depleted late CRs/nascent MRs (n > 20; Fig. 3, C and D). Thus, Anillin is required for extrusion, although we note that this may reflect a direct requirement for Anillin in the extrusion process, an indirect consequence of the requirement for Anillin in complete CR closure (Kechad et al., 2012), or both.

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