Cytokinesis In Fission Yeast Research Articles

Fission yeast type 2 node proteins Blt1p and Gef2p cooperate to ensure timely completion of cytokinesis

BackgroundThe conserved NDR-family kinase Sid2p localizes to the contractile ring during fission yeast cytokinesis to promote ring constriction, septation, and completion of cell division. Previous studies have found that the Type 2 interphase node proteins Blt1p and Gef2p contribute to localization of Sid2p and its regulatory protein Mob1p at the division site. However, their relative contributions and whether they operate in the same or parallel pathways has been unclear. In this study, we quantify the respective roles of Blt1p and Gef2p in Sid2p/Mob1p recruitment and characterize the effect of single and double deletion mutants on contractile ring dynamics and completion of cell division.ResultsUsing quantitative confocal fluorescence microscopy, we measured Sid2p and Mob1p recruitment to the division site in blt1∆, gef2∆, and blt1∆/gef2∆ mutant cells. We observed an equivalent decrease in Sid2p/Mob1p localization for both single and double mutants. Though assembly of the contractile ring is normal in these mutants, the reduction in Sid2p/Mob1p at the division site delayed the onset of contractile ring constriction and completion of division. We quantified localization of Blt1p and Gef2p at the medial cortex throughout the cell cycle and found that Blt1p localization to interphase nodes and the contractile ring is independent of Gef2p. However, Gef2p localization to the contractile ring is decreased in blt1∆ mutants.ConclusionsBlt1p and Gef2p work in the same pathway, rather than in parallel, to localize the NDR-family kinase Sid2p and its regulatory partner Mob1p to the division site, thereby promoting timely completion of cell division. Future studies are necessary to understand how additional fission yeast cytokinesis proteins work with these Type 2 interphase node components to promote Sid2p/Mob1p recruitment.

Paxillin-Mediated Recruitment of Calcineurin to the Contractile Ring Is Required for the Correct Progression of Cytokinesis in Fission Yeast

Paxillin is a scaffold protein that participates in focal adhesion signaling in mammalian cells. Fission yeast paxillin ortholog, Pxl1, is required for contractile actomyosin ring (CAR) integrity and collaborates with the β-glucan synthase Bgs1 in septum formation. We show here that Pxl1's main function is to recruit calcineurin (CN) phosphatase to the actomyosin ring; and thus the absence of either Pxl1 or calcineurin causes similar cytokinesis defects. In turn, CN participates in the dephosphorylation of the Cdc15 F-BAR protein, which recruits and concentrates Pxl1 at the CAR. Our findings suggest the existence of a positive feedback loop between Pxl1 and CN and establish that Pxl1 is a crucial component of the CN signaling pathway during cytokinesis.

Involvement of the septation initiation network in events during cytokinesis in fission yeast.

The septation initiation network (SIN), comprising a GTPase and a cascade of three protein kinases, regulates cell division in fission yeast Schizosaccharomyces pombe, but questions remain about its influence on cytokinesis. Here, we made quantitative measurements of the numbers of Cdc7p kinase molecules (a marker for SIN activity) on spindle pole bodies (SPBs), and on the timing of assembly, maturation and constriction of contractile rings via six different proteins tagged with fluorescent proteins. When SIN activity is low in spg1-106 mutant cells at 32°C, cytokinetic nodes formed contractile rings ∼3 min slower than wild-type cells. During the maturation period, these rings maintained normal levels of the myosin-II mEGFP-Myo2p but accumulated less of the F-BAR protein Cdc15p-GFP than in wild-type cells. The Cdc15p-GFP fluorescence then disintegrated into spots as mEGFP-Myo2p dissociated slowly. Some rings started to constrict at the normal time, but most failed to complete constriction. When high SIN activity persists far longer than normal on both SPBs in cdc16-116 mutant cells at 32°C, contractile rings assembled and constricted normally, but disassembled slowly, delaying cell separation.

Roles of the fission yeast UNC-13/Munc13 protein Ync13 in late stages of cytokinesis

Cytokinesis is a complicated yet conserved step of the cell-division cycle that requires the coordination of multiple proteins and cellular processes. Here we describe a previously uncharacterized protein, Ync13, and its roles during fission yeast cytokinesis. Ync13 is a member of the UNC-13/Munc13 protein family, whose animal homologues are essential priming factors for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly during exocytosis in various cell types, but no roles in cytokinesis have been reported. We find that Ync13 binds to lipids in vitro and dynamically localizes to the plasma membrane at cell tips during interphase and at the division site during cytokinesis. Deletion of Ync13 leads to defective septation and exocytosis, uneven distribution of cell-wall enzymes and components of cell-wall integrity pathway along the division site and massive cell lysis during cell separation. Interestingly, loss of Ync13 compromises endocytic site selection at the division plane. Collectively, we find that Ync13 has a novel function as an UNC-13/Munc13 protein in coordinating exocytosis, endocytosis, and cell-wall integrity during fission yeast cytokinesis.

A Hippo Pathway-Related GCK Controls Both Sexual and Vegetative Developmental Processes in the Fungus Sordaria macrospora.

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is conserved from yeast to human, and regulates a variety of key biological processes. In animals, this complex consists of the scaffold protein striatin, the protein phosphatase 2A, and kinases, such as germinal center kinase (GCK) III and GCKIV family members, as well as other associated proteins. The STRIPAK complex was identified as a negative regulator of the Hippo pathway, a large eukaryotic signaling network with a core composed of a GCK and a nuclear Dbf2-related kinase. The signaling architecture of the Hippo core resembles the fungal septation initiation network (SIN) that regulates cytokinesis in fission yeast as well as septation in filamentous fungi. In the filamentous model fungus Sordaria macrospora, core components of the STRIPAK complex have been functionally described and the striatin homolog PRO11 has been shown to interact with the GCK SmKIN3. However, the exact role of SmKIN3 in fungal development has not yet been fully elucidated. Here, we provide comprehensive genetic and functional analysis of SmKIN3 from S. macrospora. Using deletion mutants and site-directed mutagenesis, along with phenotypic and phylogenetic analysis, we provide compelling evidence that SmKIN3 is involved in fruiting body formation, hyphal fusion, and septation. Strains carrying the ATP-binding mutant SmKIN3K39R, as well as a double-deletion strain lacking SmKIN3 and the core STRIPAK subunit PRO11, also revealed severe developmental defects. Collectively, this study suggests that SmKIN3 links both the SIN and STRIPAK complex, thereby regulating multiple key cellular processes.

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.pombe)is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

Sbg1 Is a Novel Regulator for the Localization of the β-Glucan Synthase Bgs1 in Fission Yeast

Glucan synthases synthesize glucans, complex polysaccharides that are the major components in fungal cell walls and division septa. Studying regulation of glucan synthases is important as they are essential for fungal cell survival and thus popular targets for anti-fungal drugs. Linear 1,3-β-glucan is the main component of primary septum and is synthesized by the conserved β-glucan synthase Bgs1 in fission yeast cytokinesis. It is known that Rho1 GTPase regulates Bgs1 catalytic activity and the F-BAR protein Cdc15 plays a role in Bgs1 delivery to the plasma membrane. Here we characterize a novel protein Sbg1 that is present in a complex with Bgs1 and regulates its protein levels and localization. Sbg1 is essential for contractile-ring constriction and septum formation during cytokinesis. Sbg1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Bgs1 is less stable and/or mis-targeted to vacuoles in sbg1 mutants. Moreover, Sbg1 plays an earlier and more important role in Bgs1 trafficking and localization than Cdc15. Together, our data reveal a new mode of regulation for the essential β-glucan synthase Bgs1 by the novel protein Sbg1.

Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis.

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

Nuclear displacement and fluorescence recovery after photobleaching (FRAP) assays to study division site placement and cytokinesis in fission yeast.

Cytokinesis is an essential cellular event that completes the cell division cycle. It begins with the assembly of an actomyosin contractile ring that undergoes constriction concomitant with the septum formation to divide the cell in two. Placement of the septum at the right position is important to ensure fidelity of the division process. In fission yeast, the medially placed nucleus is a major spatial cue to position the site of division. In this chapter, we describe a simple synthetic biology-based approach to displace the nucleus and study the consequence on division site positioning. We also describe how to perform fluorescence recovery after photobleachingto follow the dynamics of cytokinetic proteins at defined time points by live-cell microscopy.

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis.

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.

The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

Measurements of Myosin-II Motor Activity During Cytokinesis in Fission Yeast.

Fission yeast myosin-II (Myo2p) represents the critical actin-based motor protein that drives actomyosin ring assembly and constriction during cytokinesis. We detail three different methods to measure Myo2p motor function. Actin-activated ATPases provide a readout of actomyosin ATPase motor activity in a bulk assay; actin filament motility assays reveal the speed and efficiency of myosin-driven actin filament gliding (when motors are anchored); myosin-bead motility assays reveal the speed and efficiency of myosin ensembles traveling along actin filaments (when actin is anchored). Collectively, these methods allow us to combine the standard in vivo approaches common to fission yeast with in vitro biochemical methods to learn more about the mechanistic action of myosin-II during cytokinesis.

An Extended, Boolean Model of the Septation Initiation Network in S.Pombe Provides Insights into Its Regulation.

Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

Dynamic Actomyosin Network Morphology in 3D Model of Cytokinetic Ring Assembly

At the onset of cytokinesis in fission yeast (a model organism for studying mitosis), the locations of actin filament nucleation by formins shift from the cell tips to the cell middle. These actin filaments are captured by myosin motors bound to medial cortical nodes and bundled by crosslinking proteins, in order to form a ring. The myosin motors exert forces on the actin filaments, resulting in net pulling of nodes and filaments towards the cell equator, while cross-linking interactions help align actin filaments into a single bundle. We used these mechanisms in a 3D computational model of ring assembly, with semiflexible actin filaments growing from formins at the cell tips and at the medial cell cortex, capturing and pulling of filaments by nodes, and cross-linking among filaments through attractive interactions. The model was used to predict profiles of actin density at the cortex, morphologies of condensing node-filament networks, including the transition from actin cables to ring in different conditions of myosin activity and strength of crosslinking. Results show that cross-linking interactions can lead to confinement of actin filaments that grow from medial locations of the cell cortex. 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. The presence of transient nonmedial cables linking the medial region to the cell tips can further modify these boundaries. 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.

Coarse-Grained Simulations Reveal Mechanisms of Fission Yeast Cytokinesis

Cytokinesis in fission yeast requires the presence of the actomyosin ring that constricts the membrane to divide the cell. Despite lots of effort, the molecular mechanism by which the ring tension is created remains poorly understood. This is largely due to the lack of structural details of of the ring, e.g., how actin filaments (F-actin) are arranged and what myosin conformations exist. As several models exist we decided to develop a computational approach to test whether these models are biophysically reasonable when simulated. Starting with a very simple model in which only actin filaments and bipolar myosin molecules are present we showed that myosin could slide F-actin but contraction did not occur. Addition of actin crosslinkers then helped contract the ring which in turn could pull down a coarse-grained membrane added to the system later. By tuning parameters and properties of the ring's components to match with data from electron cryotomography, we showed that coarse-grained simulations could help reveal the mechanism for the ring constriction.

Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

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.

The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis.

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1-Rho5) and seven Rho GEFs (Scd1, Rgf1-Rgf3, and Gef1-Gef3). The GEFs for Rho2-Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors--glucanases Eng1 and Agn1--are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.

INSIGHTS INTO THE MECHANICS OF CYTOKINETIC RING ASSEMBLY USING 3D MODELING.

During fission yeast cytokinesis, actin filaments nucleated by cortical formin Cdc12 are captured by myosin motors bound to a band of cortical nodes. The myosin motors exert forces that pull nodes together into a contractile ring. Cross-linking interactions help align actin filaments and nodes into a single bundle. Mutations in the myosin motor domain and changes in the concentration of cross-linkers alpha-actinin and fimbrin alter the morphology of the condensing network, leading to clumps, rings or extended meshworks. How the contractile tension developing during ring formation depends on the interplay between network morphology, myosin motor activity, cross-linking and actin filament turnover remains to be elucidated. We addressed this question using a 3D computational model in which semiflexible actin filaments (represented as beads connected by springs) grow from formins, can be captured by myosin in neighboring nodes, and get cross-linked with one another through an attractive interaction. We identify regimes of tension generation between connected nodes under a wide set of conditions regarding myosin dynamics and strength of cross-linking between actin filaments. We find conditions that maximize circumferential tension, correlate them with network morphology and propose experiments to test these predictions. This work addresses "Morphogenesis of soft and living matter" using computational modeling to simulate cytokinetic ring assembly from the key molecular mechanisms of viscoelastic cross-linked actin networks that include active molecular motors.

Fission yeast profilin is tailored to facilitate actin assembly by the cytokinesis formin Cdc12.

The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.