The contractile ring (CR) consists of bundled actin filaments and myosin II; however, the actin‐bundling factor remains elusive. We show that the fission yeast Schizosaccharomyces pombe IQGAP Rng2 is involved in the generation of CR F‐actin and required for its arrangement into a ring. An N‐terminal fragment of Rng2 is necessary for the function of Rng2 and is localized to CR F‐actin. In vitro the fragment promotes actin polymerization and forms linear arrays of F‐actin, which are resistant to the depolymerization induced by the actin‐depolymerizing factor Adf1. Our findings indicate that Rng2 is involved in the generation of CR F‐actin and simultaneously bundles the filaments and regulates its dynamics by counteracting the effects of Adf1, thus enabling the reconstruction of CR F‐actin bundles, which provides an insight into the physical properties of the building blocks that comprise the CR.

Actin‐depolymerizing factor (ADF)/cofilin is widely expressed in eukaryotes and plays a central role in reorganizing the actin cytoskeleton by disassembling actin filaments. The ADF‐homologous domain (ADF‐H) is conserved in several other actin‐modulating proteins such as twinfilin, Abp1/drebrin, and coactosin. Although these proteins interact with actin via ADF‐H, their effects on actin are not identical to each other. Here, we report a novel ADF/cofilin‐super family protein, Gmf1 (Glia maturation factor‐like protein 1), from the fission yeast Schizosaccharomyces pombe. Gmf1 is a component of actin patches, which are located on the cell cortex and required for endocytosis, and may be involved in the control of the disassembly of actin patches since its overexpression diminishes them. We provide evidence that Gmf1 binds weakly if at all to actin, but it associates with actin‐related protein (Arp) 2/3 complex and suppresses its functions such as the promotion of actin polymerization and branching filaments. Importantly, Arp2/3 complex‐suppressing activity is conserved among GMF‐family proteins from other organisms. Given the functional plasticity of ADF‐H, GMF‐family proteins possibly have changed their target from conventional actin to Arps through molecular evolution. © 2010 Wiley‐Liss, Inc.

Cytokinesis is the final stage of the cell cycle, and ensures completion of both genome segregation and organelle distribution to the daughter cells. Cytokinesis requires the cell to solve a spatial problem (to divide in the correct place, orthogonally to the plane of chromosome segregation) and a temporal problem (to coordinate cytokinesis with mitosis). Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk of tumor formation [Fujiwara et al., 2005 (Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D. 2005. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437:1043–1047); reviewed by Ganem et al., 2007 (Ganem NJ, Storchova Z, Pellman D. 2007. Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 17:157–162.)]. Asymmetric cytokinesis, which permits the generation of two daughter cells that differ in their shape, size and properties, is important both during development, and for cellular homeostasis in multicellular organisms [reviewed by Li, 2007 (Li R. 2007. Cytokinesis in development and disease: variations on a common theme. Cell Mol Life Sci 64:3044–3058)]. The principal focus of this review will be the mechanisms of cytokinesis in the mitotic cycle of the yeast Schizosaccharomyces pombe. This simple model has contributed significantly to our understanding of how the cell cycle is regulated, and serves as an excellent model for studying aspects of cytokinesis. Here we will discuss the state of our knowledge of how the contractile ring is assembled and disassembled, how it contracts, and what we know of the regulatory mechanisms that control these events and assure their coordination with chromosome segregation. © 2011 Wiley-Liss, Inc.

Adenosine triphosphate (ATP) is a main metabolite essential for all living organisms. However, our understanding of ATP dynamics within a single living cell is very limited. Here, we optimized the ATP-biosensor QUEEN and monitored the dynamics of ATP with good spatial and temporal resolution in living yeasts. We found stable maintenance of ATP concentration in wild-type yeasts, regardless of carbon sources or cell cycle stages, suggesting that mechanism exists to maintain ATP at a specific concentration. We further found that ATP concentration is not necessarily an indicator of metabolic activity, as there is no clear correlation between ATP level and growth rates. During fission yeast meiosis, we found a reduction in ATP levels, suggesting that ATP homeostasis is controlled by differentiation. The use of QUEEN in yeasts offers an easy and reliable assay for ATP dynamicity and will answer several unaddressed questions about cellular metabolism in eukaryotes.

The actomyosin-based contractile ring, which assembles at the cell equator, maintains its circularity during cytokinesis in many eukaryotic cells, ensuring its efficient constriction. Although consistent maintenance of the ring is one of the mechanisms underpinning cytokinesis, it has not yet been fully addressed. We here investigated the roles of fission yeast myosin-II proteins [Myo2 and Myo3 (also known as Myp2)] in ring maintenance during cytokinesis, with a focus on Myo3. A sitedirected mutational analysis showed that the motor properties of Myo3 were involved in its accumulation in the contractile ring. The assembled ring was often deformed and not properly maintained under conditions in which the activities of myosin-II proteins localizing to the contractile ring were decreased, leading to inefficient cell division. Moreover, Myo3 appeared to form motile clusters on the ring. We propose that large assemblies of myosin-II proteins consolidate the contractile ring by continuously binding to F-actin in the ring, thereby contributing to its maintenance.

The fission yeast Schizosaccharomyces pombe serves as a model system for studying role of actin cytoskeleton, since it has simple actin cytoskeletons and is genetically tractable. In contrast, biochemical approaches using this organism are still developing; fission yeast actin has so far not been isolated in its native form and characterized, and therefore, biochemical assays of fission yeast actin-binding proteins (ABPs) or myosin have been performed using rabbit skeletal muscle actin that may interact with the fission yeast ABPs in a manner different from fission yeast actin. Here, we report a novel method for isolating functionally active actin from fission yeast cells. The highly purified fission yeast actin polymerized with kinetics somewhat different from those of muscle actin and forms filaments that are structurally indistinguishable from skeletal muscle actin filaments. The fission yeast actin was a significantly weaker activator of Mg 2؉ -ATPase of HMM of skeletal muscle myosin than muscle actin. The fission yeast profilin Cdc3 suppressed polymerization of fission yeast actin more effectively than that of muscle actin and showed an affinity for fission yeast actin higher than for muscle actin. The establishment of purification of fission yeast actin will enable reconstruction of physiologically relevant interactions between the actin and fission yeast ABPs or myosins and contribute to clarification of function of actin cytoskeleton in various cellular activities.The actin cytoskeleton is involved in many cellular motile processes, such as muscle contraction, migration of the cell, cytoplasmic streaming, organelle positioning, cell morphogenesis, and cytokinesis. In many cases, the actin cytoskeleton is dynamic and is constructed and destroyed during the cell cycle or in response to extracellular stimuli. The dynamic nature is important for functions of the actin cytoskeleton; many actin cytoskeletons are formed only when they are necessary for specific cellular activities to be exercised. For example, the contractile ring forms in the equatorial cortex of the cell only during cytokinesis, constricts the cell into two, and is no longer present when cytokinesis is completed. The dynamic nature is mainly attributed to a couple of factors. One is the intrinsic feature of actin itself (i.e. it polymerizes into filaments and depolymerizes responding to environmental conditions). The others are regulation of the assembly feature of actin by actinmodulating proteins and formation of three-dimensional structures of actin filaments by various actin-cross-linking proteins, activities of both of which may be further controlled by upstream signaling pathways. Therefore, analyses of both assembly properties of actin and actions of various actin-binding proteins are requisite for understanding the dynamic nature of each actin cytoskeleton.The fission yeast Schizosaccharomyces pombe serves as a good model system for studying the actin cytoskeletal organization, since the cells have only three distinct F-actin structur...

During cytokinesis in many eukaryotic cells, myosin-II concentrates at the equatorial cortex with actin filaments (F-actin) and is supposed to generate forces to divide the cell into two, which is called the contractile ring (CR) hypothesis. Several lines of evidence indicate that the myosin-II is recruited independently of F-actin and interacts specifically with the equatorial F-actin. Molecular details of these mechanisms are still unknown. We used the fission yeast Schizosaccharomyces pombe to investigate the regulation of myosin-II localization. We demonstrate that the CR myosin-II was composed of F-actin-dependent and -independent fractions by simultaneously observing F-actin and myosin. The F-actin-independent fraction was visualized as cortical dots in the absence of F-actin. IQGAP Rng2, an indispensable element of CR, was implicated in maintenance of the F-actin-independent fraction of myosin-II, whereas anillin Mid1 was required for assembly but not for maintenance of the fraction. In the CR of the rng2 mutant, myosin-II was less concentrated, unstable, and nonhomogeneous, which often resulted in cytokinesis failure. These results suggest that Rng2 tethers myosin-II to the cortex along the CR independently of F-actin to provide a sufficient concentration. The robust localization of myosin-II would ensure successful cytokinesis.

Kynurenic acid (KA) is a tryptophan (Trp) metabolite that is synthesised in a branch of kynurenine (KYN) pathway. KYN aminotransferase (KAT) catalyses deamination of KYN, yielding KA. Although KA synthesis is evolutionarily conserved from bacteria to humans, the cellular benefits of synthesising KA are unclear. In this study, we constructed a KAT-null yeast mutant defective in KA synthesis to clarify the cellular function of KA. Amino acid sequence analysis and LC/MS quantification of KA revealed that Aro8 and Aro9 are the major KATs. KA was significantly decreased in the aro8Δ aro9Δ double mutant. We found that aro8Δ aro9Δ cells did not exhibit obvious defects in growth or oxidative stress response when proper amounts of amino acids are supplied in the media. We further found that aro8Δ aro9Δ cells were sensitive to excess Trp. The Trp sensitivity was not rescued by addition of KA, suggesting that Trp sensitivity is not due to the loss of KA. In conclusion, we propose that KAT activity is required for detoxification of Trp by converting it to the less toxic KA.