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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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Removal of Anillin from the nascent MR is mediated via its C-terminal domains, whereas retention requires its N-terminal domains. (A) Domain organization of Anillin and truncation mutant analyses for their localization to the nascent MR, their ability to be extruded and shed, and their retention at the mature MR. Myo, myosin; Act, actin; NTD, N-terminal domain; Sep, septin; C, C terminus; N, N terminus. (B) Time-lapse sequence of a cell coexpressing Anillin-ΔC-GFP and Anillin-ΔN+CD-mCh. Open arrowheads mark shed material, and closed arrowheads mark the mature MR. Separated channels and magnified images of the boxed regions are shown at the bottom. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 5.
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fig4: Removal of Anillin from the nascent MR is mediated via its C-terminal domains, whereas retention requires its N-terminal domains. (A) Domain organization of Anillin and truncation mutant analyses for their localization to the nascent MR, their ability to be extruded and shed, and their retention at the mature MR. Myo, myosin; Act, actin; NTD, N-terminal domain; Sep, septin; C, C terminus; N, N terminus. (B) Time-lapse sequence of a cell coexpressing Anillin-ΔC-GFP and Anillin-ΔN+CD-mCh. Open arrowheads mark shed material, and closed arrowheads mark the mature MR. Separated channels and magnified images of the boxed regions are shown at the bottom. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 5.

Mentions: Anillin is a large multidomain protein (Fig. 4 A) that can bind to many other cytokinesis proteins (D’Avino, 2009; Piekny and Maddox, 2010). We analyzed truncation mutants lacking domains to determine which mediate its retention and removal at the nascent MR. Anillin-Δactin-binding and -bundling domain (ActBD)-GFP, which lacks the ActBD identified by Field and Alberts (1995), was no longer recruited to the actin-rich cortex in mitosis, similar to the behavior of full-length Anillin-GFP in Latrunculin A–treated cells (Hickson and O’Farrell, 2008b). However, Anillin-ΔActBD-GFP was still recruited to the furrow, nascent MR, and mature MR and exhibited dramatic extrusion and shedding (Fig. S3 A). Anillin-Δmyosin-binding domain (MyoBD)-GFP, which lacks the region adjacent to the ActBD that is homologous to the Xenopus laevis myosin II–binding domain (Straight et al., 2005), still localized to the cortex at metaphase, indicating a functional ActBD, and to the CR and nascent MR during cytokinesis (Fig. S3 B). It was also readily extruded and shed and localized to mature MRs, although these appeared smaller than MRs containing full-length Anillin-GFP. Anillin-ΔN, in which the entire N terminus was deleted, leaving only the Anillin homology domain (AHD) and Pleckstrin homology (PH) domain (PHD), was still extruded and shed. Constructs expressing only the AHD or PHD were poorly recruited and showed no evidence of shedding, and deletion of either domain blocked extrusion and shedding. Thus, the C-terminal AHD and PHD are each required and together sufficient for extrusion and shedding.


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)

Removal of Anillin from the nascent MR is mediated via its C-terminal domains, whereas retention requires its N-terminal domains. (A) Domain organization of Anillin and truncation mutant analyses for their localization to the nascent MR, their ability to be extruded and shed, and their retention at the mature MR. Myo, myosin; Act, actin; NTD, N-terminal domain; Sep, septin; C, C terminus; N, N terminus. (B) Time-lapse sequence of a cell coexpressing Anillin-ΔC-GFP and Anillin-ΔN+CD-mCh. Open arrowheads mark shed material, and closed arrowheads mark the mature MR. Separated channels and magnified images of the boxed regions are shown at the bottom. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 5.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Removal of Anillin from the nascent MR is mediated via its C-terminal domains, whereas retention requires its N-terminal domains. (A) Domain organization of Anillin and truncation mutant analyses for their localization to the nascent MR, their ability to be extruded and shed, and their retention at the mature MR. Myo, myosin; Act, actin; NTD, N-terminal domain; Sep, septin; C, C terminus; N, N terminus. (B) Time-lapse sequence of a cell coexpressing Anillin-ΔC-GFP and Anillin-ΔN+CD-mCh. Open arrowheads mark shed material, and closed arrowheads mark the mature MR. Separated channels and magnified images of the boxed regions are shown at the bottom. Times are given in hours, minutes, and seconds. Bars, 5 µm. See also Video 5.
Mentions: Anillin is a large multidomain protein (Fig. 4 A) that can bind to many other cytokinesis proteins (D’Avino, 2009; Piekny and Maddox, 2010). We analyzed truncation mutants lacking domains to determine which mediate its retention and removal at the nascent MR. Anillin-Δactin-binding and -bundling domain (ActBD)-GFP, which lacks the ActBD identified by Field and Alberts (1995), was no longer recruited to the actin-rich cortex in mitosis, similar to the behavior of full-length Anillin-GFP in Latrunculin A–treated cells (Hickson and O’Farrell, 2008b). However, Anillin-ΔActBD-GFP was still recruited to the furrow, nascent MR, and mature MR and exhibited dramatic extrusion and shedding (Fig. S3 A). Anillin-Δmyosin-binding domain (MyoBD)-GFP, which lacks the region adjacent to the ActBD that is homologous to the Xenopus laevis myosin II–binding domain (Straight et al., 2005), still localized to the cortex at metaphase, indicating a functional ActBD, and to the CR and nascent MR during cytokinesis (Fig. S3 B). It was also readily extruded and shed and localized to mature MRs, although these appeared smaller than MRs containing full-length Anillin-GFP. Anillin-ΔN, in which the entire N terminus was deleted, leaving only the Anillin homology domain (AHD) and Pleckstrin homology (PH) domain (PHD), was still extruded and shed. Constructs expressing only the AHD or PHD were poorly recruited and showed no evidence of shedding, and deletion of either domain blocked extrusion and shedding. Thus, the C-terminal AHD and PHD are each required and together sufficient for extrusion and shedding.

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