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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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Maturation of the MR is accompanied by both removal and retention of Anillin. (A) Time-lapse sequence of a cell expressing Anillin-GFP and mCh-tubulin (40× objective). Arrowheads mark the mature MR before and after abscission MR release. (B–D) Time-lapse sequence of cells expressing Anillin-GFP and mCh-tubulin (not depicted) acquired with 40× objective, showing extrusion and shedding (B and C) and internalization (D). Boxed regions in B and D are shown magnified at the bottom; dashed lines in C mark the cell boundary; arrowheads mark shed material. (E) Relative Anillin-GFP fluorescence (sum intensity) at nascent MRs measured from the end of furrowing (means ± SD; n = 20). (F) FRAP experiments of nascent and mature MRs, showing images acquired immediately before (prebleach), after (bleach), and 4 min after high-intensity illumination of the outlined region of interest (63× objective and 2 × 2 camera binning). (G) The percent recovery of GFP fluorescence within 4 min of bleaching is shown for MRs of different ages (n = 9–17 each, means ± SD). P-value is for an unpaired t test. Times are hours, minutes, and seconds from the end of furrowing. Bars, 5 µm. See also Videos 1 and 2.
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fig1: Maturation of the MR is accompanied by both removal and retention of Anillin. (A) Time-lapse sequence of a cell expressing Anillin-GFP and mCh-tubulin (40× objective). Arrowheads mark the mature MR before and after abscission MR release. (B–D) Time-lapse sequence of cells expressing Anillin-GFP and mCh-tubulin (not depicted) acquired with 40× objective, showing extrusion and shedding (B and C) and internalization (D). Boxed regions in B and D are shown magnified at the bottom; dashed lines in C mark the cell boundary; arrowheads mark shed material. (E) Relative Anillin-GFP fluorescence (sum intensity) at nascent MRs measured from the end of furrowing (means ± SD; n = 20). (F) FRAP experiments of nascent and mature MRs, showing images acquired immediately before (prebleach), after (bleach), and 4 min after high-intensity illumination of the outlined region of interest (63× objective and 2 × 2 camera binning). (G) The percent recovery of GFP fluorescence within 4 min of bleaching is shown for MRs of different ages (n = 9–17 each, means ± SD). P-value is for an unpaired t test. Times are hours, minutes, and seconds from the end of furrowing. Bars, 5 µm. See also Videos 1 and 2.

Mentions: To better define the process of MR formation and maturation, we have monitored the localization of Anillin-GFP (Hickson and O’Farrell, 2008b) using time-lapse spinning-disc confocal microscopy. Anillin-GFP localized to the CR and MR throughout cytokinesis and to the MR remnant that remained associated with one of the sister cells after abscission (Fig. 1 A). Induced expression of Anillin-GFP under the control of the metallothionein promoter resulted in a fourfold overexpression of Anillin (Fig. S1, A–C). This fully rescued for loss of endogenous Anillin (Hickson and O’Farrell, 2008b) and had no consequence on the duration of furrowing or the timing of abscission when compared with other markers, such as GFP-tubulin or the myosin regulatory light chain (MRLC) Spaghetti squash–GFP (myosin-GFP; unpublished data). However, during formation of the MR, we observed a gradual thinning of the MR structure that was unexpectedly accompanied by extrusion and internalization of Anillin-GFP (Fig. 1, A–C). Extrusion originated from blebs or tubules that formed either at late stages of furrowing (Fig. 1 B and Video 1) or at the nascent MR soon after furrowing (Fig. 1 C). The extruded material persisted for several minutes and in some cases was clearly shed completely from the cell (Fig. 1, B and C, arrowheads). During internalization, Anillin-containing structures budded from the cytokinetic apparatus and were internalized into the cytoplasm as punctate vesicular structures (Fig. 1 E and Video 2). At the nascent MR, Anillin-FP sometimes labeled plasma membrane folds that had been gathered up during CR closure (Fig. S1 E). Such plasma membrane folds were also evident by EM (Fig. S1 F). Mature MRs, however, were more uniform in shape, had more closely opposed plasma membranes, and exhibited a double ring ultrastructure with a more electron-dense outer layer and a less dense inner layer (Fig. S1 F), similar to that described for intercellular canals in Drosophila melanogaster embryos (Haglund et al., 2011). The extruded structures labeled with a plasma membrane marker, myristoylated palmitoylated–GFP (myrpalm-GFP; Fig. S1), although this revealed many additional plasma membrane protrusions associated with the nascent MR that did not contain Anillin–fluorescent protein (FP; Fig. S1 G). These membrane protrusions accumulated during furrowing but then gradually dissipated during MR maturation, indicating that excess plasma membrane is gathered by the CR and then removed from the nascent MR (Fig. S1 H).


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)

Maturation of the MR is accompanied by both removal and retention of Anillin. (A) Time-lapse sequence of a cell expressing Anillin-GFP and mCh-tubulin (40× objective). Arrowheads mark the mature MR before and after abscission MR release. (B–D) Time-lapse sequence of cells expressing Anillin-GFP and mCh-tubulin (not depicted) acquired with 40× objective, showing extrusion and shedding (B and C) and internalization (D). Boxed regions in B and D are shown magnified at the bottom; dashed lines in C mark the cell boundary; arrowheads mark shed material. (E) Relative Anillin-GFP fluorescence (sum intensity) at nascent MRs measured from the end of furrowing (means ± SD; n = 20). (F) FRAP experiments of nascent and mature MRs, showing images acquired immediately before (prebleach), after (bleach), and 4 min after high-intensity illumination of the outlined region of interest (63× objective and 2 × 2 camera binning). (G) The percent recovery of GFP fluorescence within 4 min of bleaching is shown for MRs of different ages (n = 9–17 each, means ± SD). P-value is for an unpaired t test. Times are hours, minutes, and seconds from the end of furrowing. Bars, 5 µm. See also Videos 1 and 2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Maturation of the MR is accompanied by both removal and retention of Anillin. (A) Time-lapse sequence of a cell expressing Anillin-GFP and mCh-tubulin (40× objective). Arrowheads mark the mature MR before and after abscission MR release. (B–D) Time-lapse sequence of cells expressing Anillin-GFP and mCh-tubulin (not depicted) acquired with 40× objective, showing extrusion and shedding (B and C) and internalization (D). Boxed regions in B and D are shown magnified at the bottom; dashed lines in C mark the cell boundary; arrowheads mark shed material. (E) Relative Anillin-GFP fluorescence (sum intensity) at nascent MRs measured from the end of furrowing (means ± SD; n = 20). (F) FRAP experiments of nascent and mature MRs, showing images acquired immediately before (prebleach), after (bleach), and 4 min after high-intensity illumination of the outlined region of interest (63× objective and 2 × 2 camera binning). (G) The percent recovery of GFP fluorescence within 4 min of bleaching is shown for MRs of different ages (n = 9–17 each, means ± SD). P-value is for an unpaired t test. Times are hours, minutes, and seconds from the end of furrowing. Bars, 5 µm. See also Videos 1 and 2.
Mentions: To better define the process of MR formation and maturation, we have monitored the localization of Anillin-GFP (Hickson and O’Farrell, 2008b) using time-lapse spinning-disc confocal microscopy. Anillin-GFP localized to the CR and MR throughout cytokinesis and to the MR remnant that remained associated with one of the sister cells after abscission (Fig. 1 A). Induced expression of Anillin-GFP under the control of the metallothionein promoter resulted in a fourfold overexpression of Anillin (Fig. S1, A–C). This fully rescued for loss of endogenous Anillin (Hickson and O’Farrell, 2008b) and had no consequence on the duration of furrowing or the timing of abscission when compared with other markers, such as GFP-tubulin or the myosin regulatory light chain (MRLC) Spaghetti squash–GFP (myosin-GFP; unpublished data). However, during formation of the MR, we observed a gradual thinning of the MR structure that was unexpectedly accompanied by extrusion and internalization of Anillin-GFP (Fig. 1, A–C). Extrusion originated from blebs or tubules that formed either at late stages of furrowing (Fig. 1 B and Video 1) or at the nascent MR soon after furrowing (Fig. 1 C). The extruded material persisted for several minutes and in some cases was clearly shed completely from the cell (Fig. 1, B and C, arrowheads). During internalization, Anillin-containing structures budded from the cytokinetic apparatus and were internalized into the cytoplasm as punctate vesicular structures (Fig. 1 E and Video 2). At the nascent MR, Anillin-FP sometimes labeled plasma membrane folds that had been gathered up during CR closure (Fig. S1 E). Such plasma membrane folds were also evident by EM (Fig. S1 F). Mature MRs, however, were more uniform in shape, had more closely opposed plasma membranes, and exhibited a double ring ultrastructure with a more electron-dense outer layer and a less dense inner layer (Fig. S1 F), similar to that described for intercellular canals in Drosophila melanogaster embryos (Haglund et al., 2011). The extruded structures labeled with a plasma membrane marker, myristoylated palmitoylated–GFP (myrpalm-GFP; Fig. S1), although this revealed many additional plasma membrane protrusions associated with the nascent MR that did not contain Anillin–fluorescent protein (FP; Fig. S1 G). These membrane protrusions accumulated during furrowing but then gradually dissipated during MR maturation, indicating that excess plasma membrane is gathered by the CR and then removed from the nascent MR (Fig. S1 H).

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