Cytokinesis In Yeast Research Articles

The Myosin-V Myo51 and Alpha-Actinin Ain1p Cooperate during Contractile Ring Assembly and Disassembly in Fission Yeast Cytokinesis.

Cytokinesis is driven in part by the constriction of a ring of actin filaments, myosin motors and other proteins. In fission yeast, three myosins contribute to cytokinesis including a Myosin-V Myo51. As Myosin-Vs typically carry cargo along actin filaments, the role of Myo51 in cytokinesis remains unclear. The previous work suggests that Myo51 may crosslink actin filaments. We hypothesized that if Myo51 crosslinks actin filaments, cells carrying double deletions of ain1, which encodes the crosslinker alpha-actinin, and myo51 (∆ain1 ∆myo51 cells) will exhibit more severe cytokinesis phenotypes than cells with the single ∆ain1 mutation. Contrary to our expectations, we found that the loss of Myo51 in ∆ain1 cells partially rescued the severity of the node clumping phenotype measured in ∆ain1 cells. Furthermore, we describe a normal process of contractile ring "shedding", the appearance of fragments of ring material extending away from the contractile ring along the ingressing septum that occurs in the second half of constriction. We measured that ∆ain1 ∆myo51 cells exhibit premature and exaggerated shedding. Our work suggests that Myo51 is not a simple actin filament crosslinker. Instead, a role in effective node motion better recapitulates its function during ring assembly and disassembly.

Septin Organization and Dynamics for Budding Yeast Cytokinesis.

Cytokinesis, the process by which the cytoplasm divides to generate two daughter cells after mitosis, is a crucial stage of the cell cycle. Successful cytokinesis must be coordinated with chromosome segregation and requires the fine orchestration of several processes, such as constriction of the actomyosin ring, membrane reorganization, and, in fungi, cell wall deposition. In Saccharomyces cerevisiae, commonly known as budding yeast, septins play a pivotal role in the control of cytokinesis by assisting the assembly of the cytokinetic machinery at the division site and controlling its activity. Yeast septins form a collar at the division site that undergoes major dynamic transitions during the cell cycle. This review discusses the functions of septins in yeast cytokinesis, their regulation and the implications of their dynamic remodelling for cell division.

Septins function in exocytosis via physical interactions with the exocyst complex in fission yeast cytokinesis.

Septins can function as scaffolds for protein recruitment, membrane-bound diffusion barriers, or membrane curvature sensors. Septins are important for cytokinesis, but their exact roles are still obscure. In fission yeast, four septins (Spn1 to Spn4) accumulate at the rim of the division plane as rings. The octameric exocyst complex, which tethers exocytic vesicles to the plasma membrane, exhibits a similar localization and is essential for plasma membrane deposition during cytokinesis. Without septins, the exocyst spreads across the division plane but absent from the rim during septum formation. These results suggest that septins and the exocyst physically interact for proper localization. Indeed, we predicted six pairs of direct interactions between septin and exocyst subunits by AlphaFold2 ColabFold, most of them are confirmed by co-immunoprecipitation and yeast two-hybrid assays. Exocyst mislocalization results in mistargeting of secretory vesicles and their cargos, which leads to cell-separation delay in septin mutants. Our results indicate that septins guide the targeting of exocyst complex on the plasma membrane for vesicle tethering during cytokinesis through direct physical interactions.

PP2A-B56 regulates Mid1 protein levels for proper cytokinesis in fission yeast.

Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In the fission yeast S. pombe, the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that par1Δ cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide toward cell tips. These par1Δ mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses par1Δ cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.

Processes Controlling the Contractile Ring during Cytokinesis in Fission Yeast, Including the Role of ESCRT Proteins.

Cytokinesis, as the last stage of the cell division cycle, is a tightly controlled process amongst all eukaryotes, with defective division leading to severe cellular consequences and implicated in serious human diseases and conditions such as cancer. Both mammalian cells and the fission yeast Schizosaccharomyces pombe use binary fission to divide into two equally sized daughter cells. Similar to mammalian cells, in S. pombe, cytokinetic division is driven by the assembly of an actomyosin contractile ring (ACR) at the cell equator between the two cell tips. The ACR is composed of a complex network of membrane scaffold proteins, actin filaments, myosin motors and other cytokinesis regulators. The contraction of the ACR leads to the formation of a cleavage furrow which is severed by the endosomal sorting complex required for transport (ESCRT) proteins, leading to the final cell separation during the last stage of cytokinesis, the abscission. This review describes recent findings defining the two phases of cytokinesis in S. pombe: ACR assembly and constriction, and their coordination with septation. In summary, we provide an overview of the current understanding of the mechanisms regulating ACR-mediated cytokinesis in S. pombe and emphasize a potential role of ESCRT proteins in this process.

The Roles of Septins in Regulating Fission Yeast Cytokinesis.

Cytokinesis is required to separate two daughter cells at the end of mitosis, and septins play crucial roles in many aspects of cytokinesis. While septins have been intensively studied in many model organisms, including the budding yeast Saccharomyces cerevisiae, septins have been relatively less characterized in the fission yeast Schizosaccharomyces pombe, which has proven to be an excellent model organism for studying fundamental cell biology. In this review, we summarize the findings of septins made in fission yeasts mainly from four aspects: the domain structure of septins, the localization of septins during the cell cycle, the roles of septins in regulating cytokinesis, and the regulatory proteins of septins.

Meiosis II spindle disassembly requires two distinct pathways.

During exit from meiosis II, cells undergo several structural rearrangements, including disassembly of the meiosis II spindles and cytokinesis. Each of these changes is regulated to ensure that they occur at the proper time. Previous studies have demonstrated that both SPS1, which encodes a STE20-family GCKIII kinase, and AMA1, which encodes a meiosis-specific activator of the Anaphase Promoting Complex, are required for both meiosis II spindle disassembly and cytokinesis in the budding yeast Saccharomyces cerevisiae. We examine the relationship between meiosis II spindle disassembly and cytokinesis and find that the meiosis II spindle disassembly failure in sps1Δ and ama1∆ cells is not the cause of the cytokinesis defect. We also see that the spindle disassembly defects in sps1Δ and ama1∆ cells are phenotypically distinct. We examined known microtubule-associated proteins Ase1, Cin8, and Bim1, and found that AMA1 is required for the proper loss of Ase1 and Cin8 on meiosis II spindles while SPS1 is required for Bim1 loss in meiosis II. Taken together, these data indicate that SPS1 and AMA1 promote distinct aspects of meiosis II spindle disassembly, and that both pathways are required for the successful completion of meiosis.

Cdc42 prevents precocious Rho1 activation during cytokinesis in a Pak1-dependent manner.

During cytokinesis, a series of coordinated events partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated remains unclear. The GTPase Cdc42 promotes recruitment of certain cell wall-building enzymes whereas the GTPase Rho1 activates these enzymes. We show that Cdc42 prevents early Rho1 activation during fission yeast cytokinesis. Using an active Rho probe, we find that although the Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase, Rho1 is not activated until late anaphase, just before the onset of ring constriction. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, we provide functional and genetic evidence that Cdc42-dependent Rho1 inhibition is mediated by the Cdc42 target Pak1 kinase. Our work proposes a mechanism of Rho1 regulation by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity.

Development and Characterization of Expression Vectors for the Riboflavin Overproducing Fungus Eremothecium ashbyii using Ashbya gossypii Genes

Eremothecium ashbyii is a filamentous hemi-ascomycete fungus and a natural overproducer of riboflavin. The present study was undertaken to characterize the molecular tools constructed for the genetic manipulation of this organism based on plasmids constructed for the related organisms A. gossypii and S. cerevisiae using two candidate genes. The candidate gene, SPR3 homolog of S. cerevisiae, is known to play a role in cytokinesis in S. cerevisiae. This gene was chosen to aid in future studies on the regulation of septation and its role in the excretion of riboflavin in E. ashbyii, as yeast cytokinesis is analogous to the septation of filamentous fungi. The second candidate gene was the S. cerevisiae RAD14 homolog, which is known to play a key role in the nucleotide excision repair pathway. Reporter plasmids, constructed previously in a preliminary study with the AgSPR3-like gene and the AgRAD14-like gene fused to the LacZ reporter gene, were used in this study. These plasmids were characterized by sequencing followed by homology searches. While the former revealed homology to the S. cerevisiae septin protein family, SPR3 gene, and the Neurospora crassa CDC12 gene involved in cell cycle regulation, the latter showed homology to the S. cerevisiae HOGI gene involved in the osmotic stress response.

Membrane stretching activates calcium permeability of a putative channel Pkd2 during fission yeast cytokinesis.

Pkd2 is the fission yeast homologue of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayers of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypoosmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypoosmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 reduced the calcium spikes, accompanying cell separation and the ensuing membrane stretching, by 60%. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.

Cdc42 GTPase activating proteins Rga4 and Rga6 coordinate septum synthesis and membrane trafficking at the division plane during cytokinesis.

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4Δrga6Δ mutants, Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates primary septum formation and only certain membrane trafficking events. As cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity to allow coupling of Cdc42-independent membrane trafficking events with septum formation for proper septum morphology.

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis.

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.

Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis.

Cytokinesis by animals, fungi, and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentrations of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4900/min to a peak number of ∼198,000 followed by a loss of actin at 5400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost twofold more actin along with a proportional increase in type II myosins Myo2, Myp2, and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.

Anillin/Mid1p interacts with the ESCRT-associated protein Vps4p and mitotic kinases to regulate cytokinesis in fission yeast

ABSTRACT Cytokinesis is the final stage of the cell cycle which separates cellular constituents to produce two daughter cells. Using the fission yeast Schizosaccharomyces pombe we have investigated the role of various classes of proteins involved in this process. Central to these is anillin/Mid1p which forms a ring-like structure at the cell equator that predicts the site of cell separation through septation in fission yeast. Here we demonstrate a direct physical interaction between Mid1p and the endosomal sorting complex required for transport (ESCRT)-associated protein Vps4p, a genetic interaction of the mid1 and vps4 genes essential for cell viability, and a requirement of Vps4p for the correct cellular localization of Mid1p. Furthermore, we show that Mid1p is phosphorylated by aurora kinase, a genetic interaction of the mid1 and the aurora kinase ark1 genes is essential for cell viability, and that Ark1p is also required for the correct cellular localization of Mid1p. We mapped the sites of phosphorylation of Mid1p by human aurora A and the polo kinase Plk1 and assessed their importance in fission yeast by mutational analysis. Such analysis revealed serine residues S332, S523 and S531 to be required for Mid1p function and its interaction with Vps4p, Ark1p and Plo1p. Combined these data suggest a physical interaction between Mid1p and Vps4p important for cytokinesis, and identify phosphorylation of Mid1p by aurora and polo kinases as being significant for this process.

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.

Calcium spikes accompany cleavage furrow ingression and cell separation during fission yeast cytokinesis.

The role of calcium signaling in cytokinesis has long remained ambiguous. Past studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before cleavage furrow ingression, suggesting that these calcium transients could trigger contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. We explored cytokinetic calcium transients in the fission yeast Schizosaccharomyces pombe by adopting GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level of this model organism. We validated GCaMP as a highly sensitive calcium reporter in fission yeast, allowing us to capture calcium transients triggered by osmotic shocks. We identified a correlation between the intracellular calcium level and cell division, consistent with the existence of calcium transients during cytokinesis. Using time-lapse microscopy and quantitative image analysis, we discovered calcium spikes both at the start of cleavage furrow ingression and the end of cell separation. Inhibition of these calcium spikes slowed the furrow ingression and led to frequent lysis of daughter cells. We conclude that like the larger animal embryos, fission yeast triggers calcium transients that may play an important role in cytokinesis (197).

Polo-like kinase in trypanosomes: an odd member out of the Polo family.

Polo-like kinases (Plks) are evolutionarily conserved serine/threonine protein kinases playing crucial roles during multiple stages of mitosis and cytokinesis in yeast and animals. Plks are characterized by a unique Polo-box domain, which plays regulatory roles in controlling Plk activation, interacting with substrates and targeting Plk to specific subcellular locations. Plk activity and protein abundance are subject to temporal and spatial control through transcription, phosphorylation and proteolysis. In the early branching protists, Plk orthologues are present in some taxa, such as kinetoplastids and Giardia, but are lost in apicomplexans, such as Plasmodium. Works from characterizing a Plk orthologue in Trypanosoma brucei, a kinetoplastid protozoan, discover its essential roles in regulating the inheritance of flagellum-associated cytoskeleton and the initiation of cytokinesis, but not any stage of mitosis. These studies reveal evolutionarily conserved and species-specific features in the control of Plk activation, substrate recognition and protein abundance, and suggest the divergence of Plk function and regulation for specialized needs in this flagellated unicellular eukaryote.

Microtubule nucleation promoters Mto1 and Mto2 regulate cytokinesis in fission yeast.

Microtubules of the mitotic spindle direct cytokinesis in metazoans but this has not been documented in fungi. We report evidence that microtubule nucleators at the spindle pole body help coordinate cytokinetic furrow formation in fission yeast. The temperature-sensitive cps1-191 strain (Liu et al., 1999) with a D277N substitution in β-glucan synthase 1 (Cps1/Bgs1) was reported to arrest with an unconstricted contractile ring. We discovered that contractile rings in cps1-191 cells constrict slowly and that an mto2S338N mutation is required with the bgs1D277Nmutation to reproduce the cps1-191 phenotype. Complexes of Mto2 and Mto1 with γ-tubulin regulate microtubule assembly. Deletion of Mto1 along with the bgs1D277N mutation also gives the cps1-191 phenotype, which is not observed in mto2S338N or mto1Δ cells expressing bgs1+. Both mto2S338N and mto1Δ cells nucleate fewer astral microtubules than normal and have higher levels of Rho1-GTP at the division site than wild-type cells. We report multiple conditions that sensitize mto1Δ and mto2S338N cells to furrow ingression phenotypes.

Roles of Mso1 and the SM protein Sec1 in efficient vesicle fusion during fission yeast cytokinesis.

Membrane trafficking during cytokinesis is essential for the delivery of membrane lipids and cargoes to the division site. However, the molecular mechanisms are still incompletely understood. In this study, we demonstrate the importance of uncharacterized fission yeast proteins Mso1 and Sec1 in membrane trafficking during cytokinesis. Fission yeast Mso1 shares homology with budding yeast Mso1 and human Mint1, proteins that interact with Sec1/Munc18 family proteins during vesicle fusion. Sec1/Munc18 proteins and their interactors are important regulators of SNARE complex formation during vesicle fusion. The roles of these proteins in vesicle trafficking during cytokinesis have been barely studied. Here, we show that fission yeast Mso1 is also a Sec1-binding protein and Mso1 and Sec1 localize to the division site interdependently during cytokinesis. The loss of Sec1 localization in mso1Δ cells results in a decrease in vesicle fusion and cytokinesis defects such as slow ring constriction, defective ring disassembly, and delayed plasma membrane closure. We also find that Mso1 and Sec1 may have functions independent of the exocyst tethering complex on the plasma membrane at the division site. Together, Mso1 and Sec1 play essential roles in regulating vesicle fusion and cargo delivery at the division site during cytokinesis.