Directionality of F-actin cables changes during the fission yeast cell cycle

Longitudinal F-actin cables are thought to be important for transporting materials for polarized cell growth in fission yeast. We show that most F-actin in the cables is oriented such that the barbed end faces the nearest cell tip during interphase; however, this directionality is reversed during mitosis. These orientations of F-actin ensure proper transport of materials to growing sites during these cell-cycle stages.     

Three-dimensional arrangement of F-actin in the contractile ring of fission yeast

The contractile ring, which is required for cytokinesis in animal and yeast cells, consists mainly of actin filaments. Here, we investigate the directionality of the filaments in fission yeast using myosin S1 decoration and electron microscopy. The contractile ring is composed of around 1,000 to 2,000 filaments each around 0.6 mum in length. During the early stages of cytokinesis, the ring consists of two semicircular populations of parallel filaments of opposite directionality. At later stages, before contraction, the ring filaments show mixed directionality. We consider that the ring is initially assembled from a single site in the division plane and that filaments subsequently rearrange before contraction initiates.     

The actomyosin ring recruits early secretory compartments to the division site in fission yeast

The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring.     

Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1-3]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4-5]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.     

Role of the protein kinase Kin1 and nuclear centering in actomyosin ring formation in fission yeast

Cytokinesis is the last step of the cell cycle, producing two daughter cells inheriting equal genetic information. This process involves the assembly of an actomyosin ring during mitosis. In the fission yeast Schizosaccharomyces pombe, cytokinesis occurs at the geometric cell centre, a position which is defined by the interphase nucleus and the anilin-related Mid1 protein. The pom1Δ, tea1Δ and tea4Δ mutants are defective in restricting Mid1 as a band around the nucleus and misplace the division site. We previously reported that inhibition of the protein kinase Kin1 promoted failure of cytokinesis in pom1Δ and tea1Δ cells but the mechanism involving Kin1 remained elusive. Here we investigated the contribution of Kin1 in cytokinesis. We show that Kin1-GFP has a dynamic cell-cycle regulated distribution. Like pom1Δ and tea1Δ, tea4Δ exhibits a strong genetic interaction with kin1Δ. Using a conditional repressible kin1 allele that only alters interphase nuclear centering, we observed that Kin1 down-regulation severely compromised actomyosin ring formation and septum synthesis in tea4Δ cells. In addition, nuclear displacement induced either by overexpression of a putative catalytically inactive Kin1 mutant, by chemically mediated microtubule depolymerization or by mutation in the par1Δ gene impaired cytokinesis in tea4Δ but not tea4+ cells. We propose that nuclear mispositioning exacerbates the tea4Δ, pom1Δ and tea1Δ cell division phenotype. Our work reveal that nuclear centering becomes essential when Pom1/Tea1/Tea4 function is compromised and that Kin1 expression level is a key regulatory element in this situation. Our results suggest the existence of distinct overlapping control mechanisms to ensure efficient cell division.     

Pxl1p, a paxillin-related protein, stabilizes the actomyosin ring during cytokinesis in fission yeast

Paxillins are a family of conserved LIM domain-containing proteins that play important roles in the function and integrity of the actin cytoskeleton. Although paxillins have been extensively characterized by cell biological and biochemical approaches, genetic studies are relatively scarce. Here, we identify and characterize a paxillin-related protein Pxl1p in the fission yeast Schizosaccharomyces pombe. Pxl1p is a component of the fission yeast actomyosin ring, a structure that is essential for cytokinesis. Cells deleted for pxl1 display a novel phenotype characterized by a splitting of the actomyosin ring in late anaphase, leading to the formation of two rings of which only one undergoes constriction. In addition, the rate of actomyosin ring constriction is slower in the absence of Pxl1p. pxl1Delta mutants display strong genetic interactions with mutants defective in IQGAP-related protein Rng2p and mutants defective in components of the fission yeast type II myosin machinery. Collectively, these results suggest that Pxl1p might cooperate with type II myosin and Rng2p-IQGAP to regulate actomyosin ring constriction as well as to maintain its integrity during constriction.     

Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast

Cell division in a number of eukaryotes, including the fission yeast Schizosaccharomyces pombe, is achieved through a medially placed actomyosin-based contractile ring. Although several components of the actomyosin ring have been identified, the mechanisms regulating ring assembly are still not understood. Here, we show by biochemical and mutational studies that the S.pombe actomyosin ring component Cdc4p is a light chain associated with Myo2p, a myosin II heavy chain. Localization of Myo2p to the medial ring depended on Cdc4p function, whereas localization of Cdc4p at the division site was independent of Myo2p. Interestingly, the actin-binding and motor domains of Myo2p are not required for its accumulation at the division site although the motor activity of Myo2p is essential for assembly of a normal actomyosin ring. The initial assembly of Myo2p and Cdc4p at the division site requires a functional F-actin cytoskeleton. Once established, however, F-actin is not required for the maintenance of Cdc4p and Myo2p medial rings, suggesting that the attachment of Cdc4p and Myo2p to the division site involves proteins other than actin itself.     

Cytokinesis and the contractile ring in fission yeast

The fission yeast Schizosaccharomyces pombe provides a genetic model system for the study of cytokinesis. As in many eukaryotes, cell division in the fission yeast requires an actin-myosin-based contractile ring. Numerous components of the contractile ring that function in ring assembly, positioning and contraction have been characterized. Many of these proteins are evolutionarily conserved, suggesting that common molecular mechanisms may govern aspects of eukaryotic cell division. Recent advances in the assembly and placement of the contractile ring are discussed. In particular, major findings have been made in the characterization of myosins in cytokinesis, and in how the cell division site may be positioned by the nucleus.     

Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis

Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in budding yeast, actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to different ploidies.     

Contractile ring formation in Xenopus egg and fission yeast

How actin filaments (F-actin) and myosin II (myosin) assemble to form the contractile ring was investigated with fission yeast and Xenopus egg. In fission yeast cells, an aster-like structure composed of F-actin cables is formed at the medial cortex of the cell during prophase to metaphase, and a single F-actin cable(s) extends from this structure, which seems to be a structural basis of the contractile ring. In early mitosis, myosin localizes as dots in the medial cortex independently of F-actin. Then they fuse with each other and are packed into a thin contractile ring. At the growing ends of the cleavage furrow of Xenopus eggs, F-actin at first assembles to form patches. Next they fuse with each other to form short F-actin bundles. The short bundles then form long bundles. Myosin seems to be transported by the cortical movement to the growing end and assembles there as spots earlier than F-actin. Actin polymerization into the patches is likely to occur after accumulation of myosin. The myosin spots and the F-actin patches are simultaneously reorganized to form the contractile ring bundles. The idea that a Ca signal triggers cleavage furrow formation was tested with Xenopus eggs during the first cleavage. We could not detect any Ca signals such as a Ca wave, Ca puffs or even Ca blips at the growing end of the cleavage furrow. Furthermore, cleavages are not affected by Ca-chelators injected into the eggs at concentrations sufficient to suppress the Ca waves. Thus we conclude that formation of the contractile ring is not induced by a Ca signal at the growing end of the cleavage furrow.     

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP₂-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.     

Role of the two type II myosins,Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeast

The formation and contraction of a cytokinetic actomyosin ring (CAR) is essential for the execution of cytokinesis in fission yeast. Unlike most organisms in which its composition has been investigated, the fission yeast CAR contains two type II myosins encoded by the genes myo2(+) and myp2(+). myo2(+) is an essential gene whilst myp2(+) is dispensable under normal growth conditions. Myo2 is hence the major contractile protein of the CAR whilst Myp2 plays a more subtle and, as yet, incompletely documented role. Using a fission yeast strain in which the chromosomal copy of the myo2(+) gene is fused to the gene encoding green fluorescent protein (GFP), we analysed CAR formation and function in the presence and absence of Myp2. No change in the rate of CAR contraction was observed when Myp2 was absent although the CAR persisted longer in the contracted state and was occasionally observed to split into two discrete rings. This was also observed in myp2Delta cells following actin depolymerisation with latrunculin. CAR contraction in the absence of Myp2 was completely abolished in the presence of elevated levels of chloride ions. Thus, Myp2 appears to contribute to the stability of the CAR, in particular at a late stage of CAR contraction, and to be a component of the signalling pathway that regulates cytokinesis in response to elevated levels of chloride. To determine whether the presence of two type II myosins was a feature of cytokinesis in other fungi that divide by septation, we searched the genomes of two filamentous fungi, Aspergillus fumigatus and Neurospora crassa, for myosin genes. As in fission yeast, both A. fumigatus and N. crassa contained myosins of classes I, II, and V. Unlike fission yeast, both contained a single type II myosin gene that, on the basis of its tail structure, was more reminiscent of Myp2 than Myo2. The significance of these observations to our understanding of septum to formation and cleavage is discussed.     

Cytokinesis in budding yeast: the relationship between actomyosin ring function and septum formation

Cytokinesis in budding yeast is accomplished by the concerted action of actomyosin ring function and septum formation. The actomyosin ring is not essential for cell viability, but it is required for efficient cell division. Deletion of the actomyosin ring results in abnormal septum formation, and a delay in cytokinesis and cell separation. In contrast, septum formation is essential for cell viability. Block of septum formation prevents the contraction, but not the formation of the actomyosin ring. Here we review and provide additional evidence that defines the functional and molecular relationship between actomyosin ring function and septum formation.     

A Highlights from MBoC Selection: Myosin Vs organize actin cables in fission yeast

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.     

Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast

Cytokinesis in many eukaryotes depends on the function of an actomyosin contractile ring. The mechanisms regulating assembly and positioning of this ring are not fully understood. The fission yeast Schizosaccharomyces pombe divides using an actomyosin ring and is an attractive organism for the study of cytokinesis. Recent studies in S. pombe (Wu, J.Q., V. Sirotkin, D.R. Kovar, M. Lord, C.C. Beltzner, J.R. Kuhn, and T.D. Pollard. 2006. J. Cell Biol. 174:391–402; Vavylonis, D., J.Q. Wu, S. Hao, B. O'Shaughnessy, and T.D. Pollard. 2008. Science. 319:97–100) have suggested that the assembly of the actomyosin ring is initiated from a series of cortical nodes containing several components of this ring. These studies have proposed that actomyosin interactions bring together the cortical nodes to form a compacted ring structure. In this study, we test this model in cells that are unable to assemble cortical nodes. Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings. Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.     

Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.     

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis

The functions of the actin-myosin–based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells.     

Detection and characterization of a ring chromosome in the fission yeast Schizosaccharomyces pombe.

NotI and SfiI genomic restriction maps were used to detect and characterize a ring chromosome II in a Schizosaccharomyces pombe strain with a meiotic defect on chromosome II. The ring chromosome was formed by an intrachromosomal fusion near, or at, the very ends of chromosome II.