Contractile Ring Assembly

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Contractile-Ring Assembly in Fission Yeast Cytokinesis:Recent Advances and New Perspectives

I-Ju Lee,1,2

Valerie C. Coffman,1

and Jian-Qiu Wu1,3

*1Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 2 Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, Ohio 3 Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio

Received 27 April 2012; Accepted 16 July 2012Monitoring Editor: Douglas Robinson

The fission yeast Schizosaccharomyces pombe is anexcellent model organism to study cytokinesis. Here, we review recent advances on contractile-ring assembly

in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contract-ile ring. IQGAP Rng2 and myosin essential light chainCdc4 are recruited by the anillin-like protein Mid1,followed by the addition of other cytokinesis nodeproteins. Mid1 localization on the plasma membrane isstabilized by interphase node proteins. Second, we dis-cuss proteins and processes that contribute to thesearch, capture, pull, and release mechanism of con-tractile-ring assembly. Actin filaments nucleated by for-min Cdc12, the motor activity of myosin-II, thestiffness of the actin network, and severing of actin

filaments by cofilin all play essential roles in contract-ile-ring assembly. Finally, we discuss the Mid1-independent pathway for ring assembly, and thepossible mechanisms underlying the ring maturationand constriction. Collectively, we provide an overview of the current understanding of contractile-ring assem-bly and uncover future directions in studying cytokine-sis in fission yeast. VC 2012 Wiley Periodicals, Inc

Key Words: anillin, contractile ring, S. pombe, nodes,search, capture, pull, and release model

Introduction

C ytokinesis partitions a mother cell into two daughtercells at the end of each cell cycle. Failure in cytokine-sis results in tetraploidy and contributes to tumorigenesis[Fujiwara et al., 2005; Ganem et al., 2007; Li et al.,2010; Sagona and Stenmark, 2010]. The actomyosin

contractile ring in cytokinesis is conserved from fungi tohumans [Pollard and Wu, 2010]. Research using the fis-sion yeast Schizosaccharomyces pombe as a model system

has provided novel insights into contractile-ring assembly.Briefly, contractile-ring assembly in fission yeast beginswhen Mid1, the anillin-like protein, recruits other pro-teins to assemble cytokinesis nodes [Paoletti and Chang,2000; Wu et al., 2003; Motegi et al., 2004; Laporte et al.,2011; Padmanabhan et al., 2011]. The nodes later con-dense into a compact actomyosin contractile ring througha process that has been described as a search, capture,pull, and release (SCPR) mechanism, which depends ontransient interactions between the myosin-II motors andlinear actin filaments [Vavylonis et al., 2008]. The con-tractile ring then matures by recruiting additional compo-

nents before its constriction [Wu et al., 2003]. Althoughcontractile-ring assembly can be described by a seemingly simple model, the mechanisms and regulation underlying each step of the assembly process are complex and involvemany proteins. Therefore, contractile-ring assembly in fis-sion yeast has been an active field of research. Recentstudies in fission yeast cytokinesis not only shed light onthe assembly and architecture of cytokinesis nodes, butalso elucidate several proteins’ contributions to the SCPR mechanism. Here, we review our current understanding of contractile-ring assembly, and discuss future perspectivesin investigating cytokinesis in fission yeast.

Cytokinesis Node Assembly

In fission yeast, the anillin-like protein Mid1 specifies thedivision site [Chang and Nurse, 1996; Sohrmann et al.,1996; B€ahler et al., 1998; Paoletti and Chang, 2000;Celton-Morizur et al., 2004; Daga and Chang, 2005; Almonacid et al., 2009]. In interphase, Mid1 localizes toboth the nucleus and the plasma membrane at the equatorof the cell [Sohrmann et al., 1996; B€ahler et al., 1998;Paoletti and Chang, 2000]. At the G2/M transition, Mid1is mainly concentrated on the medial cortex and joined by

*Address correspondence to: Jian-Qiu Wu, Room 615, BiologicalSciences Building, The Ohio State University, Columbus, OH43210, USA. E-mail: [email protected]

Published online 23 August 2012 in Wiley Online Library (wileyonlinelibrary.com).

REVIEW ARTICLECytoskeleton, October 2012 69:751–763 (doi: 10.1002/cm.21052)VC 2012 Wiley Periodicals, Inc.

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several other proteins to form an equatorial band of 65cortical punctate structures named cytokinesis nodes that

have a Gaussian distribution along the long axis of thecell [Paoletti and Chang, 2000; Wu et al., 2003, 2006;Vavylonis et al., 2008]. These cortical nodes later coalesceinto the contractile ring [B€ahler et al., 1998; Wu et al.,2003, 2006], suggesting that these macromolecular corti-cal complexes (on the cytoplasmic side of the plasma membrane) are precursors of the contractile ring in fissionyeast cells (Fig. 1A). Proteins in cytokinesis nodes includethe anillin-like protein Mid1 [B€ahler et al., 1998; Paolettiand Chang, 2000], the IQGAP protein Rng2 [Eng et al.,1998], the myosin-II motor (heavy chain Myo2, essentiallight chain Cdc4, and regulatory light chain Rlc1)

[McCollum et al., 1995; Kitayama et al., 1997; May et al., 1997; Naqvi et al., 1999, 2000; Bezanilla et al.,2000; Motegi et al., 2000, 2004], the F-BAR proteinCdc15 [Fankhauser et al., 1995; Carnahan and Gould,2003], and the formin Cdc12 [Chang et al., 1997; Wuet al., 2006; Coffman et al., 2009]. Their localizations tothe nodes are actin-independent [Motegi et al., 2000; Pao-letti and Chang, 2000; Wu et al., 2003, 2006]. In mid1Dcells, equatorial cytokinesis nodes do not form, and cellsare severely defective in division-site selection andcontractile-ring assembly [Wu et al., 2003, 2006]. Mid1phosphorylation by Polo kinase Plo1 is crucial for the

initiation of cytokinesis-node assembly [B€ahler et al.,1998; Almonacid et al., 2011] (for a detailed review of the regulation of Mid1 and division-site selection, see thereview by Rincon and Paoletti [2012] in this issue).

Although Rng2, Cdc4, Myo2, Rlc1, Cdc15, and Cdc12only colocalize with Mid1 beginning at G2/M, severalproteins colocalize with Mid1 on the cortex during mostof interphase [Moseley et al., 2009]. The interphase corti-cal structures that contain Mid1 and these proteins arehence named ‘‘interphase nodes,’’ which determine cellsize and mitotic entry together with Pom1 kinase [Morrellet al., 2004; Martin and Berthelot-Grosjean, 2009; Mose-ley et al., 2009; Hachet et al., 2011]. Note that in wildtype cells, the different nomenclature simply reflects thedifference of node components in regard to cell cyclestage. Here, we discuss the connections between interphaseand cytokinesis nodes and review recent advances on theassembly of cytokinesis nodes.

Cytokinesis Nodes and Interphase Nodes

Proteins in interphase nodes include three kinases Cdr2[Breeding et al., 1998; Kanoh and Russell, 1998; Morrellet al., 2004], Cdr1 [Young and Fantes, 1987; Feilotteret al., 1991], and Wee1 [Parker et al., 1991; Wu et al.,1996; Masuda et al., 2011], kinesin-like protein Klp8, theputative Rho guanine nucleotide exchange factor (GEF)Gef2, and novel protein Blt1 [Martin and Berthelot-Gros- jean, 2009; Moseley et al., 2009]. In mitosis, Klp8, Gef2,and Blt1 persist in cytokinesis nodes, but are not essentialfor contractile-ring assembly because without interphase

nodes, Mid1 can still localize to the medial cortex at theG2/M transition and assemble cytokinesis nodes inde-pendently [Almonacid et al., 2009]. Nevertheless, evidencesuggests that interphase node proteins can contribute tothe regulation of cytokinesis [Almonacid et al., 2009;Laporte et al., 2011; Ye et al., 2012].

Cdr2, the SAD/GIN4 kinase in fission yeast, is the or-ganizer of interphase nodes and involved in regulating cellsize by inhibiting Wee1 kinase [Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009]. Cdr2 is detected incondensing cytokinesis nodes but disappears from the di-vision site shortly after the assembly of a compact con-

tractile ring [Martin and Berthelot-Grosjean, 2009;Moseley et al., 2009]. Interestingly, Mid1, IQGAP Rng2,and F-BAR protein Cdc15 nodes are more dynamic inthe absence of Cdr2, indicating that interphase node pro-teins play a role in stabilizing cytokinesis nodes [Laporteet al., 2011].

Gef2, Blt1, and Klp8 stay in the contractile ring untilthe end of ring constriction [Moseley et al., 2009; Yeet al., 2012]. Although the function of Klp8 remainsunknown, strong synthetic genetic interactions in gef2 D plo1-ts18 cells and quantitative microscopy indicate thatGef2 and Polo kinase Plo1 are required together to

Fig. 1. The assembly of cytokinesis nodes and the contract-ile ring in fission yeast. ( A ) Contractile-ring assembly andcytokinesis in fission yeast. Interphase nodes are orange. Cytoki-

nesis nodes and the contractile ring are red. Nuclei are blue. Actin filaments are green. (B) The assembly hierarchy of cyto-kinesis nodes. Hypothetical protein shapes/structures used in(C) are shown close to each protein name. Both the F-BAR protein Cdc15 and the Rng2-Cdc4 module (gray box) couldrecruit the formin Cdc12. (C) The architecture of a cytokinesisnode. Yellow filaments are F-actin nucleated by Cdc12. PM,plasma membrane. (B and C) Modified from Laporte et al.[2011].

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regulate Mid1 cortical level in cytokinesis. blt1D alsoshows mild genetic interaction with plo1-ts18 , and itsinvolvement in cytokinesis is partially through Gef2 [Yeet al., 2012]. The functions of Gef2 and Blt1 in inter-phase are not clear yet, although gef2 D and blt1D cells areslightly longer than wild type cells [Moseley et al., 2009; Ye et al., 2012].

It seems surprising that the cell-size sensing machinery and the precursors of the contractile ring are colocalized,but this arrangement could promote timely assembly of the cytokinesis apparatus once a cell is committed toundergo mitosis. Nodes are not the only cortical structureobserved in S. pombe . Rga4, the Rho GTPase (guanosinetriphosphatase)-activating protein that inhibits growth oncell sides, localizes to punctate-like structures on the cor-tex [Das et al., 2007; Tatebe et al., 2008] that are distinctfrom nodes (our unpublished data). Eisosomes in fissionyeast assemble into filaments on the cortex [Kabecheet al., 2011]. Clustering of the plasma membrane compo-

nents may be the origin of the distinction between thesedifferent structures [Wachtler et al., 2003; Morrell et al.,2004; Takeda and Chang, 2005]. Whether cross-talks existbetween these structures requires further investigation.

The Assembly and Architecture of

Cytokinesis Nodes

Spindle pole bodies (SPB) are the yeast counterparts of animal centrosomes. SPB separation marks the onset of mitosis in fission yeast. All the cytokinesis node proteinsexcept Mid1 arrive at nodes shortly before or around SPB

separation [Wu et al., 2003; Laporte et al., 2011].Recently, several studies have determined the hierarchy of cytokinesis-node assembly using complementary methods[Almonacid et al., 2011; Laporte et al., 2011; Padmanab-han et al., 2011]. First, localization dependencies of cyto-kinesis proteins at nodes were determined by using temperature-sensitive mutants and germinated spores of null mutants (Fig. 1B). Second, the appearance of theproteins at nodes was imaged and quantified with hightemporal resolution. Third, their dynamics on nodes wereobtained by fluorescence recovery after photobleaching (FRAP). Fourth, Mid1 phosphorylation and the physical

interactions between Mid1 and other cytokinesis proteinswere determined. Last but not least, the architecture of nodes was revealed by a modified single-molecule high-re-solution colocalization (SHREC) technique that breaksthe diffraction limit and results in a resolution of tens of nanometers [Churchman et al., 2005; Joglekar et al.,2009]. The architecture of cytokinesis nodes obtained issummarized in Fig. 1C.

These studies highlight the importance of the IQGAPprotein Rng2 and Cdc4, which are the earliest to appearat nodes (12 min before SPB separation) after Mid1.Except Mid1, other cytokinesis node proteins are not

required for Rng2 and Cdc4 to localize. However, withoutfunctional Rng2 and Cdc4, Myo2 and Rlc1 cannot local-ize to nodes. Rng2 interacts with Mid1 in co-immunopre-cipitation (co-IP) [Laporte et al., 2011; Padmanabhanet al., 2011], and rng2-M1 (with mutations H1329L andK1366E in the region interacting with Mid1) phenocopiesmid1D [Padmanabhan et al., 2011]. The C-terminus (aa 1306–1489) of Rng2 interacts with the N-terminus (first100 aa) of Mid1 in an in vitro binding assay [Almonacidet al., 2011], suggesting that Rng2 is directly recruited by Mid1. The N-terminus (aa 1-100) of Mid1, when tar-geted to nodes, is sufficient to assemble cytokinesis nodesand the contractile ring [Lee and Wu, 2012]. Cdc4 formsa complex with Mid1 in co-IP, but it is unknown if theinteraction is direct or not. Rng2 and Cdc4 interact witheach other via the IQ motifs in Rng2 [D’Souza et al.,2001], and their localizations to cytokinesis nodes areinterdependent [Laporte et al., 2011; Padmanabhan et al.,2011]. Taken together, Rng2 and Cdc4 are more

upstream in the node-assembly pathway but whether they are recruited as a complex is unknown.

Cdc4 has two recovery rates in FRAP analysis. Although the slow recovery resembles that of Rng2, thefast recovery depends on its interaction with the IQ motif of Myo2 [Naqvi et al., 2000; D’Souza et al., 2001;Laporte et al., 2011]. Consistent with the timing of appearance (10 min before SPB separation) and thelocalization dependency, the weak interaction between en-dogenous Myo2 and Mid1 revealed by co-IP depends onfunctional Rng2 [Laporte et al., 2011]. Results fromSHREC also suggest that Myo2 is further away from the

plasma membrane compared to Mid1 and Rng2, with itsmotor head pointing into the cytoplasm and C-terminaltail folded. Rlc1, the regulatory light chain of myosin-II,displays the same timing of appearance and dynamics asMyo2, and its localization completely depends on Myo2[Laporte et al., 2011].

F-BAR protein Cdc15 arrives at nodes 5 min beforeSPB separation [Laporte et al., 2011]. The dephosphoryl-ation of Cdc15, partly regulated by the Cdc14-family phosphatase Clp1 [Trautmann et al., 2001; Wolfe andGould, 2004; Wolfe et al., 2006], is crucial for its divi-sion-site localization, conformation, and interactions

[Wachtler et al., 2006; Clifford et al., 2008; Roberts-Galbraith et al., 2010]. Prematurely dephosphorylatedCdc15 localizes to cortical nodes and is able to causemedial localization of its interacting partners in interphase[Roberts-Galbraith et al., 2010]. Some discrepancy existsregarding the relationship between Mid1, Rng2, andCdc15 (Fig. 1B). Physical interactions are reportedbetween Mid1-Cdc15 (yeast two hybrid and co-IP assays;[Laporte et al., 2011]), Mid1-Rng2 (co-IP and in vitrobinding assay using purified fragments; [Almonacid et al.,2011; Laporte et al., 2011; Padmanabhan et al., 2011]),and Cdc15-Rng2 (tandem affinity purification [TAP]- and

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co-IP; [Roberts-Galbraith et al., 2010]). On the one hand,Laporte et al. [2011] proposed that Mid1 can recruitCdc15 to cytokinesis nodes independent of the Rng2-Cdc4 module, because Cdc15 nodes are detected in rng2-D5 , rng2-346 , rng2 D, cdc4-8 , and cdc4 D mutants. On the

other hand, Padmanabhan et al. proposed that Cdc15 isdownstream of the Rng2-Cdc4 module because no Cdc15nodes are detected in the rng2-M1 mutant at the restrictivetemperature. It is possible that part of the discrepancy comes from the difference between rng2 mutants [Padma-nabhan et al., 2011]. Indeed, the total Mid1 protein levelis lower in rng2-M1 but not in rng2-D5 cells [Laporteet al., 2011; Padmanabhan et al., 2011]. Another possibil-ity is that the higher autofluorescence at the GFP (greenfluorescent protein) channel compared to the YFP (yellow fluorescent protein) channel might obscure a weak corticalCdc15 signal in rng2-M1 [Padmanabhan et al., 2011].

Cdc15 levels in the nodes have not been quantified in thecdc4 and rng2 mutants [Laporte et al., 2011], so the possi-bility remains that both Mid1 and Rng2 are involved inrecruiting Cdc15. Further studies are needed to addressthese different possibilities.

The last known component to join cytokinesis nodesbefore their condensation is the formin Cdc12, whoseinteraction with Cdc15 is well studied [Carnahan andGould, 2003; Roberts-Galbraith et al., 2010]. Surpris-ingly, it was recently found that in addition to Cdc15, theRng2-Cdc4 module can also recruit Cdc12 to cytokinesisnodes [Laporte et al., 2011] (Fig. 1B). Cdc12 is dispensa-ble for other cytokinesis node proteins to localize [Laporteet al., 2011], although its function to nucleate actin fila-ments is essential for contractile-ring assembly [Kovar et al., 2003]. Soon after Cdc12 appears at the divi-sion site, cytokinesis nodes start to condense and the

Fig. 2. The SCPR mechanism of contractile-ring assembly. ( A–D) Two nodes are pulled closer during the search (A), capture (B),pull (C), and release (D) process. Shaded areas (light blue) indicate the following events: (1) formin Cdc12 nucleates and elongatesan actin filament from profilin-bound monomers. (2) A myosin-II motor captures the actin filament. (3) Tropomyosin stabilizes theactin filament. (4) Myosin-II motor activity induced by the UCS protein Rng3 pulls two nodes closer. (5) The crosslinking by a-acti-nin resists the movement. (6) The actin filament dissociates from myosin-II. (7) The actin filament is severed by cofilin. (8) Theactin filament is capped at its barbed end by capping protein. (9) Cdc12 dissociates from the rest of the node.



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contractile ring assembles in 10 min in wild type cells at25C.

SCPR Model and Beyond

Since the proposal that the contractile ring in fission yeast

cells is assembled from the condensation of a broad bandof nodes [B€ahler et al., 1998; Wu et al., 2006], numericalsimulations and live-cell imaging of cytokinesis node pro-teins and actin filaments were integrated to describe themechanism. Monte Carlo simulations, using parametersobtained in vivo, recapitulate the condensation of nodesvia transient connections between actin filaments andneighboring nodes. Vavylonis et al. [2008] therefore pro-posed the SCPR mechanism of contractile-ring assembly:an actin filament nucleated by formin Cdc12 in one nodesearches the cortex and can be captured by the myosin-IIat another node, and the force generated by the myosin-II

motor walking on the actin filament pulls the nodes closerbefore the release of the interaction. The SCPR modeldiffers from the previously proposed spot/leading cablemodel for contractile-ring assembly in the numbers of actin-nucleation sites, orientations of actin filaments, andthe importance of myosin-II motor activity [Chang et al.,1997; Chang, 1999; Arai and Mabuchi, 2002; Carnahanand Gould, 2003; Kamasaki et al., 2007; Mishra and Oli-ferenko, 2008; Coffman et al., 2009]. Key assumptionsand predictions in the SCPR model were tested subse-quently in a number of studies (see below). Consistentwith the model, many different perturbations of contract-

ile-ring assembly result in discontinuous aggregates(clumps) on the cortex rather than a continuous ring.Meanwhile, in vivo observations have led to the refining of the model [Ojkic et al., 2011; Laporte et al., 2012].Here, we review the process of SCPR, and discuss playersthat contribute to each step of contractile-ring assembly (Fig. 2).

Search

After the nodes have matured by the recruitment of theformin Cdc12, actin polymerization is crucial for the

‘‘search’’ step. Cdc12 is the essential formin that nucleatesactin filaments at the division site [Chang et al., 1997;Kovar et al., 2003; Coffman et al., 2009]. Therefore, itsactivity and localization are critical for contractile-ring as-sembly. As previous studies suggest that the majority of actin filaments for contractile-ring assembly are nucleatedby Cdc12 at the division site [Pelham and Chang, 2002;Coffman et al., 2009], actin filaments nucleated elsewherein cells are not included in the SCPR simulation. Never-theless, current data do not exclude the possibility thatthese filaments could be incorporated into the contractilering.

The number of actin nucleation sites in the broad band of nodes determines the success and efficiency of contractile-ring assembly. Assuming there are 65 nodes in each cell,the SCPR model requires that at least 50% of them shouldcontain formins and nucleate 1–4 filaments from each nodein order for the nodes to condense into a ring in 10 min.In agreement with the model, 2–4 dimers of Cdc12 localizeto >50% of nodes right before the nodes start to condense[Coffman et al., 2009; Laporte et al., 2011]. The highnucleation efficiency (1 filament out of 3 dimers) of puri-fied Cdc12 FH1FH2 domain in vitro [Scott et al., 2011]supports both the SCPR model and the in vivo data [Coff-man et al., 2009; Laporte et al., 2011].

The orientation and elongation rate of each actin fila-ment determines its chance to encounter another node.On the cell cortex, actin filaments are randomly orientedat the beginning of node condensation [Coffman et al.,2009], as applied in the SCPR model. Further study showed that actin filaments at the division site exhibit an

average angle of 8 to the plasma membrane, possibly dueto the position and orientation of Cdc12 in nodes, affinity of actin filaments with the plasma membrane, or restric-tion by the endoplasmic reticulum [Zhang et al., 2010;Laporte et al., 2011]. This angle may ensure that the actinfilaments can be readily captured (see below). The lengthof actin filaments is controlled by actin-binding proteinsand the processivity of Cdc12. Profilin Cdc3 [Balasubra-manian et al., 1994] is required for the rapid elongationof actin filaments by Cdc12 [Kovar et al., 2003] (Fig.2A). In the presence of profilin, Cdc12 associates withelongating actin filaments processively [Kovar and Pollard,

2004] and competes with capping protein better thanother formins [Neidt et al., 2008]. Tropomyosin Cdc8[Balasubramanian et al., 1992] helps the elongation of actin filaments by inhibiting disassembly [Skau et al.,2009]. The binding of tropomyosin to actin and thus itslocalization is regulated by acetylation [Skoumpla et al.,2007; Coulton et al., 2010].

Of note, the regulation of Cdc12 activity remainslargely unknown. No Rho GTPase has been identified toactivate Cdc12, although many diaphanous-related for-mins are regulated in this way. Because cdc12 is an essen-tial gene, domain analyses were performed in the presence

of the endogenous protein, adding complexity to theinterpretation of the results [Yonetani et al., 2008; Yone-tani and Chang, 2010]. The formation of interphase ringswhen a C-terminal Cdc12 truncation is overexpressed inthe presence of endogenous Cdc12 is suggestive of someform of inhibition that acts on the long C-terminal tail of Cdc12 [Yonetani and Chang, 2010]. A formin damper orinhibitor, such as Smy1 or Bud14 in S. cerevisiae [Chesar-one et al., 2009; Chesarone-Cataldo et al., 2011], has notbeen found yet in S. pombe . The temperature-sensitivemutant cdc12-112 forms many small clumps over theequator, consistent with the results of SCPR when actin



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filaments are too short [Hachet and Simanis, 2008; Ojkicand Vavylonis, 2010]. Because deletion of capping proteinrescues the cdc12-112 phenotype, it has been suggestedthat this formin mutant lacks processivity [Kovar et al.,2005], which would result in shorter actin filaments andmake capture events less likely.

Capture

When an actin filament nucleated from a node encountersanother node (in the SCPR model, when the filamentcomes within the capture radius 100 nm from the cent-roid of the node), it might be captured by myosin-II (Fig.2B). Tropomyosin Cdc8 stabilizes actin filaments (Fig.2B) and increases the affinity of myosin-II for actin fila-ments [Stark et al., 2010]. It has been suggested that withthe motor head of myosin-II being oriented away fromthe cortex in stationary nodes, it is more likely to catchthe slightly inward-pointing actin filaments [Laporte et al.,

2011]. Increasing myosin-II concentration speeds up con-tractile-ring assembly maybe partly by increasing captureevents, or by producing more force and thus pulling thenodes together more quickly [Stark et al., 2010].

Other actin-binding proteins in nodes, such as Rng2[Eng et al., 1998], could also capture actin filaments[Takaine et al., 2009], but the interaction would notresult in shortening of the distance between nodes withoutthe myosin-II motor activity. The interaction betweenRng2 and actin filaments may generate tension and pullthem closer to the myosin motor heads and thus increasethe chance of capture by myosin-II. On the other hand,Rng2-actin interaction might interfere with the SCPR mechanism if actin filaments are stabilized but notdirected to myosin-II. A Rng2 truncation lacking theactin-binding Calponin Homology Domain would behelpful for further analysis of its function and the impor-tance of its actin-binding activity. Because Rng2 is essen-tial, such a truncation may not be viable, although itwould not be expected to affect the node assembly path-way [Laporte et al., 2011].

In the original SCPR model [Vavylonis et al., 2008], actinfilaments stop growing once they are captured by neighbor-ing nodes, and the tension-induced switch-off is importantfor the mechanism. Whether actin filaments stop growing

after being captured in vivo remains untested.

Pull

A key assumption in the SCPR model is that the forcegenerated by the myosin-II motor on the captured actinfilament pulls the two nodes closer to each other. It pre-dicts that mutants with defective myosin-II motor activity cannot condense the nodes properly. Indeed, Coffmanet al. [2009] showed that when myo2-E1, a temperature-sensitive mutant with weakened myosin-II motor activity,is grown at the restrictive temperature, actin filaments are

nucleated at the division site and associated with cytokine-sis nodes, but node condensation is severely affected.Phosphorylation of the regulatory light chain was sug-gested to regulate myosin-II motor activity [Chew et al.,1998; Sanders et al., 1999; Loo and Balasubramanian,2008]. Indeed, mutating phosphorylation sites on Rlc1 toalanine results in lower Myo2 motility in vitro and a delay

in contractile-ring assembly at higher temperatures in vivo[Sladewski et al., 2009] (see below for discussion). The fis-sion yeast UCS protein Rng3 [Wong et al., 2000] acti-vates the motor activity of myosin-II (Fig. 2C) [Lord andPollard, 2004; Lord et al., 2008]. When the temperature-sensitive rng3-65 cells are grown at the restrictive tempera-ture, the movement of nodes is minimal; when the cellsare shifted back to the permissive temperature, node con-densation is recovered [Coffman et al., 2009]. Thus, myo-sin-II motor activity is essential for node condensationinto the contractile ring.

If actin filaments are crosslinked into a network (Fig.

2C), then the pulling forces of myosin motors will be dis-tributed to all the other nodes that are connected togetherthrough actin filaments. Although this could help coordi-nate node condensation, crosslinking may also bundleactin filaments along unproductive directions. Recentstudies highlight the importance of actin-crosslinking pro-teins in cytokinesis. When semiflexible actin filaments arecrosslinked, the stiffness of the network increases, thusmaking node condensation more difficult. a-Actinin formsa homodimer via its spectrin repeats and bundles actin fil-aments [Xu et al., 1998; Djinovic-Carugo et al., 1999]. Inmammalian cells, the crosslinking by a-actinin is required

for structural support of the actin network during cytoki-nesis, because the depletion of a-actinin results in a sud-den collapse of the equatorial cortical network [Mukhina et al., 2007].

Ain1, the a-actinin in S. pombe , also localizes to thecontractile ring [Nakano et al., 2001; Wu et al., 2001]. Although both deleting and overexpressing ain1 causedelays in contractile-ring assembly, live-cell imaging showscompletely opposite behavior of actin filaments and cyto-kinesis node proteins in these strains [Laporte et al.,2012]. In ain1D, the actin network becomes moredynamic and cytokinesis nodes usually first condense into

a clump before a contractile ring is eventually formed.This is likely due to excess net pulling force because of less resistance via crosslinking. When excess Ain1 is pres-ent in the cell, the movement of nodes is attenuated andactin filaments form stable linear structures that may ormay not assemble into a contractile ring. A balancebetween myosin pulling force and the damping effect of crosslinkers is reestablished when Myo2 is slightly overex-pressed in these cells, which leads to successful contractile-ring assembly [Laporte et al., 2012]. These results suggestthat the extent of crosslinking is critical for proper con-tractile-ring assembly. In addition to Ain1, Rng2 has also



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been shown to bundle actin filaments [Takaine et al.,2009]. In contrast, Cdc12 has no bundling activity [Scottet al., 2011]. Recent evidence suggests that actin cross-linker fimbrin Fim1 functions as a subsidiary to Ain1 dur-ing contractile-ring assembly, although Ain1 has a moreprominent role during node condensation, probably dueto the difference in the geometry/distance of the cross-linked actin filaments [Laporte et al., 2012].

Release

Permanent interactions between nodes result in a series of clumps instead of a ring at the equator in the simulationsof the SCPR model. Therefore, the release of interactionsis critical for contractile-ring assembly. In the SCPR model, the release between two nodes happens with a con-stant rate [Vavylonis et al., 2008]. In vivo, several factorscould contribute to the release of the interaction between

two nodes (Fig. 2D). First, Myosin-II could dissociatefrom the captured actin filament. Myosin-II is a motorwith low duty ratio and


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stiffness of the actin network restrains the orientation of nodes and the direction of actin filament elongation atlater stages is unknown. The arrangement of F-actin inthe contractile ring of wild type cells revealed by myosinS1 decoration and electron microscopy indicates that in a full-sized contractile ring, two semicircular populations of parallel filaments with opposite orientations exist during early anaphase B; in a constricting ring, filaments with op-posite orientations are mixed homogenously throughoutthe ring [Kamasaki et al., 2007]. Investigating the orienta-tions of F-actin in cells during late stages of node conden-sation, just before the formation of a compact ring, willbe helpful to compare actin directionality at early versuslate stages of node condensation. Although the architec-ture of the node before condensation indicates that themyosin-II motor head points toward the cytoplasm anddoes not support the formation of antiparallel myosinminifilaments, whether a change of node architecture may allow the minifilaments to form at later stages of node

condensation remains unclear.

Mid1-Independent Contractile-Ring Assembly

In mid1D cells, there are no equatorial cytokinesis nodes,and contractile-ring assembly is severely impaired [Wuet al., 2003]. However, most mid1D cells are viable, indi-cating the existence of other mechanisms to assemble theessential contractile ring. Some mid1D cells can slowly make a randomly positioned and randomly oriented ring from linear structures consisting of Rng2, Myo2, Rlc1,Cdc12, actin, and other cytokinesis proteins [Wu et al.,

2003; Hachet and Simanis, 2008; Huang et al., 2008;Coffman et al., 2009]. Recent studies suggest that theSIN pathway is involved in contractile-ring assembly incells lacking functional Mid1 [Hachet and Simanis, 2008;Huang et al., 2008; Roberts-Galbraith and Gould, 2008;Bathe and Chang, 2010] (For a detailed review of theSIN pathway, please see Johnson et al. [2012] in thisissue). Hyperactivation of the SIN pathway induces con-tractile-ring assembly in interphase [Hachet and Simanis,2008; Huang et al., 2008]. SIN-induced rings assemblefrom filamentous or linear structures that arise from ran-dom positions, resembling rings assembled in mid1D cells.

When the SCPR mechanism is not compromised, SINmainly functions in later stages of cytokinesis (see ‘‘Ring maturation and constriction’’ section). In SIN mutants, a compact ring can form but cannot mature and constrictbefore its collapse [Balasubramanian et al., 1998; Houet al., 2000; Krapp et al., 2001; Hachet and Simanis,2008]. Although contractile rings can form in mid1-6 andsid2-250 (SIN kinase) single mutants, it cannot form inthe double mutants [Hachet and Simanis, 2008], suggest-ing the existence of parallel and/or sequential pathwaysfor ring assembly in fission yeast. The Polo kinase Plo1regulates both pathways and both are important for the

formation of a functional contractile ring at the correct di-vision site [B€ahler et al., 1998; Tanaka et al., 2001; Almo-nacid et al., 2011].

The SIN-induced, Mid1-independent rings are able toconstrict more slowly than normal rings [Hachet andSimanis, 2008], allowing completion of cytokinesis insome cells. It may be that without equatorial cytokinesisnodes in mid1D cells, the filamentous or linear structurescould incorporate and spread the essential ring compo-nents and thus make a functional contractile ring [Rob-erts-Galbraith and Gould, 2008; Bathe and Chang,2010]. However, the molecular mechanism of Mid1-inde-pendent ring assembly remains obscure. In addition, it isunknown why SIN-induced rings are less homogenousand constrict slower [Hachet and Simanis, 2008].

Taken together, contractile-ring assembly in wild typefission yeast can be successfully modeled using the SCPR mechanism, and the Mid1-independent ring assembly depends on the SIN pathway. In the SCPR model, each

parameter consists of numerous events in the cells andinvolves many proteins. Contractile-ring assembly requiresmultiple rounds of SCPR. Therefore, defects in one stepmight lead to severe consequences. However, many pro-teins are involved in more than one step in the process.Thus, the in vivo results obtained are not always easy tointerpret. In vitro assays have provided many insights, butin vivo analyses with high spatial and temporal resolutionare required to distinguish different possibilities. It will beof interest to further test the assumptions and predictionsof the SCPR model, and characterize proteins and proc-esses that contribute to each step. In addition, integrating

results from both node-dependent and Mid1-independentcontractile-ring assembly will be a challenge in the future.

Ring Maturation and Constriction

In wild type cells, the condensation of nodes results in a compact ring without lagging nodes at 10 min afterSPB separation. The diameter of the ring stays constantfor 25 min before constriction begins [Wu et al., 2003].During this stage, the contractile ring matures by concen-trating many additional proteins to the ring and/or to thedivision site adjacent to the ring, including additional F-

BAR protein Cdc15, capping protein, the unconventionalmyosin-II heavy chain Myp2 [Bezanilla et al., 1997,2000; Wu et al., 2003; Kovar et al., 2005], Rho GTPasesand their regulators [Mutoh et al., 2005; Nakano et al.,2005; Rincon et al., 2007; Wu et al., 2010; Arasada andPollard, 2011], Arp2/3 complex and its activators [Carna-han and Gould, 2003; Takeda and Chang, 2005; Wuet al., 2006], septins [Wu et al., 2003; An et al., 2004],and many other proteins [Pollard and Wu, 2010].

Compared to cytokinesis-node and contractile-ring as-sembly, much less is known about ring maturation andconstriction. Nevertheless, many studies indicate that the



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F-BAR protein Cdc15 and the SIN pathway play impor-tant roles during this stage. The number of Cdc15 mole-cules in the contractile ring increases 10-fold during ring maturation [Wu and Pollard, 2005]. Without functionalCdc15, a compact contractile ring can assemble but fallsapart [Fankhauser et al., 1995; Balasubramanian et al.,1998; Wachtler et al., 2006; Hachet and Simanis, 2008].The regulation of ring integrity is mediated at least partly through the SH3 domain of Cdc15. It interacts with theC2 domain protein Fic1 and the paxillin Pxl1, two of theproteins that appear at the division site during ring matu-ration and are involved in maintaining ring integrity [Geand Balasubramanian, 2008; Pinar et al., 2008; Roberts-Galbraith et al., 2009]. Imp2, another F-BAR protein infission yeast, cooperates with Cdc15 during this process[Roberts-Galbraith et al., 2009]. Failure to maintain ring integrity is also observed in SIN mutants with reducedCdc15 recruitment to the division site [Hachet and Sima-nis, 2008].

In most mutants that exhibit a delay in contractile-ring assembly, the initiation of ring constriction is not delayed.For example in myo2-E1 cells, it takes much longer toassemble the contractile ring even at permissive tempera-ture, but once the contractile ring is formed, it only undergoes a very short ‘‘dwell time’’ before constrictionbegins [Coffman et al., 2009; Stark et al., 2010]. In con-trast, in cells with two copies of myo2 , the contractile ring assembles prematurely, leaving a prolonged dwell timebefore ring constriction [Stark et al., 2010]. In mid1Dand mid1 mutants, once the SIN-dependent ring isassembled after a delay, its diameter also starts to decrease

immediately [Celton-Morizur et al., 2004; Hachet andSimanis, 2008; Huang et al., 2008]. Together, these resultssuggest that ring maturation and constriction are tightly controlled and probably regulated through a cell-cycle-de-pendent signal/mechanism. When a delay in ring assembly occurs, the ring may mature while being assembled. Therecruitment of additional ring components during ring maturation could assist the defective assembly process insome mutants. For example, the SIN-dependent pathway could recruit additional Cdc15 to complement a defectiveSCPR mechanism in several mutants [Hachet and Sima-nis, 2008], and the arrival of Myp2 may support the

eventual ring assembly observed in myo2-E1 mutants.Given that many proteins recruited in ring maturation arenecessary for ring constriction, recruitment of these pro-teins during delayed assembly could allow the contractilering to constrict at the normal time. Future studies shouldfocus on characterizing the effect of proteins recruited dur-ing ring maturation on mutant ring assembly processes.

In addition to the recruitment of more components,several lines of evidence suggest that the ring undergoesreorganization during maturation and constriction. First,the disappearance of Mid1 from the ring at the onset of ring constriction suggests that other proteins take over its

role to anchor the contractile ring to the plasma mem-brane. The postanaphase array of microtubules and b-glu-can synthase Bgs1/Cps1 contribute to the anchoring of contractile ring at the equator of the cell [Pardo andNurse, 2003]. Second, different dynamics of cytokinesisproteins in FRAP assays may suggest that the organizationof cytokinesis nodes, fully assembled rings, and constrict-ing rings are different, although variance exists in how these experiments were performed [Clifford et al., 2008; Yonetani et al., 2008; Coffman et al., 2009; Laporteet al., 2011]. The reorganization likely prepares the ring for constriction.

Myosin-II motor activity is required for both the assem-bly and constriction of the contractile ring. Because thediameter of the contractile ring does not change while thering matures, it will be interesting to investigate how themyosin-II motor activity is regulated at this time. It ispossible that after node condensation into a compact ring,the motor activity is turned off during ring maturation,

and activated again for constriction. It has been suggestedthat Rlc1, phosphorylated by Pak1, inhibits motor activity of Myo2 until ring constriction [Naqvi et al., 2000; Looand Balasubramanian, 2008]. However, an in vitro assay suggested the opposite result [Sladewski et al., 2009]. A Pak1 mutant with impaired kinase function acceleratesring constriction when anaphase progression is sloweddown in the ase1D mutant [Loo and Balasubramanian,2008], but the same phenotype was not observed withnonphosphorylatable Rlc1 in ase1þ background [Sladewskiet al., 2009]. Therefore, whether the myosin-II motor isswitched off during ring maturation remains elusive.

Alternatively, the lateral redistribution of ring compo-nents along the arc length during ring maturation, asobserved in 4D projections of GFP-Cdc15 and Rlc1-GFP[Wu et al., 2006; Hachet and Simanis, 2008], suggeststhat the myosin motor may remain active at this stage. If so, ring constriction must be prevented by other mecha-nisms so that the ring diameter remains constant during ring maturation. It has been shown that turgor pressure infission yeast cells is very high [Minc et al., 2009]. It ispossible that the force produced by active myosin-II walk-ing on actin filaments is enough to slide actin filamentslaterally along the plasma membrane during ring assembly,

but not sufficient to overcome the turgor pressure to con-strict the ring during ring maturation. It will be enlighten-ing to investigate if septum formation and membraneinsertion provide additional forces to overcome the highturgor pressure during ring constriction.

Ring constriction is regulated by the SIN pathway. SINcomponents localize to SPBs, but Sid2 kinase and itsinteracting partner Mob1 [Salimova et al., 2000] alsolocalize to the contractile ring [Sparks et al., 1999; Houet al., 2000]. Sid2 phosphorylation of Clp1 is required forthe retention of Clp1 in the cytoplasm, and the mutationof Sid2 phosphorylation sites on Clp1 causes defects in



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cytokinesis [Chen et al., 2008]. Other Sid2 substratespresent in the contractile ring remain to be identified.

Our understanding of ring maturation and constrictionis far from complete. Investigating when and how differ-ent proteins are added to the contractile ring will shedlight on the process of ring maturation. In addition, it isimportant to investigate what kind of reorganization takesplace while the ring matures. Unlike in S. cerevisiae [Young et al., 2010], the cytokinesis apparatus in S. pombe has not been successfully isolated and purified yet, and itsmore dynamic nature and bigger diameter make it chal-lenging. In contrast, quantitative live-cell imaging prom-ises to be a powerful tool in determining theconcentration and dynamics of ring components. Three-dimensional reconstitution or a system that allows thecells to be imaged vertically to overcome the poor z-reso-lution will be particularly helpful in studying the architec-ture of the ring and will potentially reveal the change of ring organization during ring constriction.

Conclusions and Perspectives

In this review, we have summarized recent advances oncontractile-ring assembly in fission yeast. Cytokinesisnodes assemble in a hierarchical order and condense intoa compact contractile ring through a process described asthe SCPR mechanism. Each step in the mechanism iscontributed by a subset of proteins that regulate myosin-IIactivity and actin dynamics. Although the mechanism isless clear, the compact ring matures by recruiting addi-tional components and undergoes remodeling before and

perhaps also during its constriction.Gaining new information to refine the established

model in fission yeast will benefit the field of cytokinesissignificantly. Although thoroughly investigating the func-tion and regulation of key players such as the anillin-likeMid1, IQGAP Rng2, myosin-II complex, the F-BAR pro-tein Cdc15, and formin Cdc12 will allow us to furtherexamine and polish the mechanism of contractile-ring as-sembly, we need to keep in mind that many other pro-teins are likely involved in this process. In fission yeast,250 different proteins localize to the division site [Mat-suyama et al., 2006], at least 130 proteins have been

reported to be involved in cytokinesis, and many of themare conserved from yeasts to humans [Pollard and Wu,2010]. It is necessary to analyze the genetic and physicalinteractions among these proteins systematically and eluci-date whether they contribute to contractile-ring assembly.

Acknowledgments

We thank Dimitrios Vavylonis for critical reading of thismanuscript and members of the Wu laboratory for helpfuldiscussions. I-J.L. and V.C.C. are supported by Pelotonia Graduate Fellowship and Elizabeth Clay Howald Presiden-tial Fellowship, respectively. The work in J.-Q.W. labora-

tory is supported by The Ohio State University andNational Institutes of Health grant R01GM086546.

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Contractile Ring Assembly