Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.     

Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast

In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in Δmid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ∼67% of cells, strands slowly close into rings that constrict without the normal ∼25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.     

UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.     

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8⁺ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.     

Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.     

IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast

Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.     

Temporal control of contractile ring assembly by Plo1 regulation of myosin II recruitment by Mid1/anillin

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes. These nodes then coalesce laterally into a functional contractile ring. Although Mid1 is present at the medial cortex throughout G2, recruitment of contractile ring components to nodes starts only at mitotic onset, indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis, and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset, coupling division plane specification to nuclear position. Here we provide evidence that Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2, and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset.     

Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis

Microscopy of fluorescent fusion proteins and genetic dependencies show that fission yeast assemble and constrict a cytokinetic contractile ring in a precisely timed, sequential order. More than 90 min prior to separation of the spindle pole bodies (SPB), the anillin-like protein (Mid1p) migrates from the nucleus and specifies a broad band of cortex around the equator as the division site. Between 10 min before and 2 min after SPB separation, conventional myosin-II (Myo2p), IQGAP (Rng2p), PCH protein (Cdc15p), and formin (Cdc12p) join the broad band independent of actin filaments. Over the subsequent 10 min prior to anaphase B, this broad band of proteins condenses into a contractile ring including actin, tropomyosin (Cdc8p), and alpha-actinin (Ain1p). During anaphase B, unconventional myosin-II (Myp2p) joins the ring followed by the septin (Spn1p). Ring contraction and disassembly begin 37 min after SPB separation. This spatial and temporal hierarchy provides the framework for analysis of molecular mechanisms.     

Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle–dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20–30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle–dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.     

Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.     

Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring

Eukaryotic cells require IQGAP family multidomain adapter proteins for cytokinesis, but many questions remain about how IQGAPs contribute to the process. Here we show that fission yeast IQGAP Rng2p is required for both the normal process of contractile ring formation from precursor nodes and an alternative mechanism by which rings form from strands of actin filaments. Our work adds to previous studies suggesting a role for Rng2p in node and ring formation. We demonstrate that Rng2p is also required for normal ring constriction and septum formation. Systematic analysis of domain-deletion mutants established how the four domains of Rng2p contribute to cytokinesis. Contrary to a previous report, the actin-binding calponin homology domain of Rng2p is not required for viability, ring formation, or ring constriction. The IQ motifs are not required for ring formation but are important for ring constriction and septum formation. The GTPase-activating protein (GAP)–related domain is required for node-based ring formation. The Rng2p C-terminal domain is the only domain essential for viability. Our studies identified several distinct functions of Rng2 at multiple stages of cytokinesis.     

An InCytes from MBC Selection: Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.     

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.     

The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis

Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.     

Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring. One of the key components of this ring is the F-actin based motor protein myosin II. The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p. Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p. In this study, we have created and characterized S. pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains. Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function. Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities. Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished. Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis. Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p. We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.     

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.     

Dynamic Network Morphology and Tension Buildup in a 3D Model of Cytokinetic Ring Assembly

During fission yeast cytokinesis, actin filaments nucleated by cortical formin Cdc12 are captured by myosin motors bound to a band of cortical nodes and bundled by cross-linking proteins. The myosin motors exert forces on the actin filaments, resulting in a net pulling of the nodes into a contractile ring, while cross-linking interactions help align actin filaments and nodes into a single bundle. We used these mechanisms in a three-dimensional computational model of contractile ring assembly, with semiflexible actin filaments growing from formins at cortical nodes, capturing of filaments by neighboring nodes, and cross-linking among filaments through attractive interactions. The model was used to predict profiles of actin filament density at the cell cortex, morphologies of condensing node-filament networks, and regimes of cortical tension by varying the node pulling force and strength of cross-linking among actin filaments. Results show that cross-linking interactions can lead to confinement of actin filaments at the simulated cortical boundary. We show that the ring-formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables. Since boundaries between regions are not sharp, transient structures that resemble clumps, stars, and meshworks can appear in the process of ring assembly. These results are consistent with prior experiments with mutations in actin-filament turnover regulators, myosin motor activity, and changes in the concentration of cross-linkers that alter the morphology of the condensing network. Transient star shapes appear in some simulations, and these morphologies offer an explanation for star structures observed in prior experimental images. Finally, we quantify tension along actin filaments and forces on nodes during ring assembly and show that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed.     

Characterization of structural and functional domains of the anillin-related protein Mid1p that contribute to cytokinesis in fission yeast

Fission yeast cells depend on the anillin-related protein Mid1p for reliable cytokinesis. Insolubility limits the purification of full-length Mid1p for biophysical analysis, and lack of knowledge about the structural domains of Mid1p limits functional analysis. We addressed these limitations by identifying in a bacterial expression screen of random Mid1p fragments five soluble segments that can be purified and one insoluble segment. Using complementation experiments in Δmid1 cells, we tested the biological functions of these six putative domains that account for full-length Mid1p. The N-terminal domain (residues 1–149) is essential for correct positioning and orientation of septa. The third domain (residues 309–452) allows the construct composed of the first three domains (residues 1-452) to form hydrodynamically well-behaved octamers. Constructs consisting of residues 1–452 or 1–578 carry out most functions of full-length Mid1p, including concentration at the equatorial cortex in nodes that accumulate myosin-II and other contractile ring proteins during mitosis. However, cells depending on these constructs without the insoluble domain (residues 579–797) form equatorially located rings slowly from strands rather than by direct condensation of nodes. We conclude that residues 1–578 assemble node components myosin-II, Rng2p, and Cdc15p, and the insoluble domain facilitates the normal, efficient condensation of nodes into rings.