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.

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One fundamental problem in cytokinesis is how the plane of cell division is established. In this review, we describe our studies on searching for "signals" that position the cell division plane, using fission yeast Schizosaccharomyces pombe. First, we take a genetic approach to determine how the nucleus may position the contractile ring in fission yeast. mid1p appears to link the position of the ring with the nuclear position, as it is required for proper placement of the contractile ring and is localized in a band at the cell surface overlying the nucleus. Second, we study how microtubules may function in the establishment of cell polarity at the cell tips. tea1p may be deposited on the cell surface by microtubules and function to recruit proteins involved in making actin structures. These studies suggest how microtubules may direct the assembly of the contractile ring in animal cells.

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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.

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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, IQ1 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.

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Recent studies have demonstrated that vesicle transport to cleavage furrow is indispensable for cytokinesis. Some animal and plant cells form distinct structures during cell division known as central spindle and phragmoplast, respectively. Several essential factors involved in the vesicle transport have been isolated so far. SNARE proteins and molecular motors play a central role in this process. For future research of cytokinesis, it is important to investigate these factors as well as cytoskeletal components of the contractile ring in detail. This review focuses on the molecular mechanism of targeted vesicle transport in cytokinesis.

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Dictyostelium is one of the model systems of choice for studying the cytokinesis of animal-type cells. Two types of cytokinesis mutants have been used to identify proteins involved in the cytokinesis of Dictyostelium: (1) type I, the mutant cells grow on substrates to produce giant multinucleate cells; (2) type II, the mutant cells divide nearly normally on substrates, but are unable to divide at all and get highly multinucleate in suspension culture. These two mutant types might correspond to the myosin II-independent and myosin II-including cytokinesis mechanisms, respectively.

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The ability of Dictyostelium cells to divide without myosin II in a cell cycle-coupled manner has opened two questions about the mechanism of cleavage furrow ingression. First, are there other possible functions for myosin II in this process except for generating contraction of the furrow by a sliding filament mechanism? Second, what could be an alternative mechanical basis for the furrowing? Using aberrant changes of the cell shape and anomalous localization of the actin-binding protein cortexillin I during asymmetric cytokinesis in myosin II-deficient cells as clues, it is proposed that myosin II filaments act as a mechanical lens in cytokinesis. The mechanical lens serves to focus the forces that induce the furrowing to the center of the midzone, a cortical region where cortexillins are enriched in dividing cells. Additionally, continual disassembly of a filamentous actin meshwork at the midzone is a prerequisite for normal ingression of the cleavage furrow and a successful cytokinesis. If this process is interrupted, as it occurs in cells that lack cortexillins, an overassembly of filamentous actin at the midzone obstructs the normal cleavage. Disassembly of the crosslinked actin network can generate entropic contractile forces in the cortex, and may be considered as an alternative mechanism for driving ingression of the cleavage furrow. Instead of invoking different types of cytokinesis that operate under attached and unattached conditions in Dictyostelium, it is anticipated that these cells use a universal multifaceted mechanism to divide, which is only moderately sensitive to elimination of its constituent mechanical processes.

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Dictyostelium discoideum is a unique experimental organism which allows genetic analysis of the mechanism of cytokinesis of the animal type, and a number of mutations which affect cytokinesis in one way or other have been identified. Myosin II filaments accumulate in the equatorial region, and myosin II-null cells cannot divide in suspension, indicating that active, myosin II-dependent constriction of the cleavage furrow contributes to bisection of the cell. We refer to this method of cytokinesis as cytokinesis A. On substrates, however, myosin II-null cells divide efficiently in a cell cycle-coupled manner. This adhesion-dependent but myosin II-independent division method, which we termed cytokinesis B, is carried out by a pathway that is genetically distinct from that of cytokinesis A. Morphological analyses suggested that cytokinesis B is driven by radial traction forces generated along polar peripheries, which indirectly cause furrow ingression. Identification of two redundant pathways have allowed us to search genes involved in either pathway by mutagenizing cells which are already defective in one of the pathways. This approach enabled us to identify a number of novel cytokinesis-related genes, as well as to reclassify known genes as cytokinesis-related.

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In the molecular mechanism of division plane determination and contractile ring formation, Tetrahymena 85kDa protein (p85) is localized to the presumptive division plane before the formation of the contractile ring. p85 directly interacts with Tetrahymena calmodulin (CaM) in a Ca²⁺-dependent manner, and p85 and CaM colocalize in the division furrow. A Ca²⁺/CaM inhibitor N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl (W7) inhibits the direct interaction between p85 and Ca²⁺/CaM. W7 also inhibits the localization of p85 and CaM to the division plane, and the formation of the contractile ring and division furrow. In addition, p85 binds to G-actin in a Ca²⁺/CaM dependent manner, but does not bind F-actin. Tetrahymena profilin is localized to division furrow and binds Tetrahymena elongation factor-1 α (EF-1α). EF-1α, which induces bundling of Tetrahymena F-actin, is also localized to the division furrow during cytokinesis. The evidence also indicates that Ca²⁺/CaM inhibits the F-actin-bundling activity of EF-1α, and that EF-1α and CaM colocalize in the division furrow. In this review, we propose that the Ca²⁺/CaM signal and its target protein p85 cooperatively regulate the determination of the division plane and the initiation of the contractile ring formation, and that profilin and a Ca²⁺/CaM-sensitive actin-bundling protein, EF-1α, play pivotal roles in regulating the organization of the contractile ring microfilaments.

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Genetic and molecular studies in the nematode Caenorhabditis elegans have identified multiple essential pathways that regulate and execute cytokinesis in early embryonic cells. These pathways influence both the microfilament cytoskeleton and the microtubule cytoskeleton. Microfilaments are enriched throughout the cell cortex at all times during the cell cycle in embryonic cells. Cortical microfilaments are required for multiple processes in embryonic cells, including polar body extrusion during meiosis, anterior-posterior axis specification by the sperm-donated microtubule-organizing center, and cytokinesis during mitosis. In addition to contractile apparatus proteins that are required positively for cleavage furrow ingression, the Nedd8 ubiquitin-like protein modification pathway negatively regulates contractile forces outside the cleavage furrow during cytokinesis. Another pathway that acts positively during cytokinesis involves the mitotic spindle. The central spindle, where anti-parallel non-kinetochore microtubules overlap and are cross-linked, is required for a late step in cytokinesis, and other pathway(s) involved in membrane addition during cytokinesis may also require the central spindle. The amenability of C. elegans to classical genetics, the ease of reducing gene function with RNA interference, the completion of the genome sequence, and the availability of transgenic GFP fusion proteins that render the cytoskeleton fluorescent, all serve to make the early worm embryo an especially promising system for further advances in the identification of cytokinesis pathways, and in defining their interactions.

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Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other.

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Several G proteins of the Rho family have been shown to be required for cytokinesis. The activity of these proteins is regulated by GTP exchange factors (GEFs), which stimulate GDP/GTP exchange, and by GTPase activating proteins (GAPs), which suppress activity by stimulating the intrinsic GTPase activity. The role of Rho family members during cytokinesis is likely to be determined by their spatial and temporal interactions with these factors. Here we focus on the role of the pebble (pbl) gene of Drosophila melanogaster, a RhoGEF that is required for cytokinesis. We summarise the evidence that the primary target of PBL is Rho1 and describe genetic approaches to elucidating the function of PBL and identifying other components of the PBL-activated Rho signalling pathway.

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In order to understand the mechanism of unequal division, polar body formation was investigated using the oocytes of the starfish, Asterina pectinifera. Cortical actin filaments were quantitatively measured after staining the maturing oocytes with fluorescently labeled phalloidin using a computer and image-processing software. Before polar body formation, at first the actin filaments at the animal pole decreased and subsequently the animal pole bulged. On the other hand, actin filaments surrounding the animal pole increased gradually and made a cleavage furrow around the animal pole as the bulge grew. Then the furrow ingressed and finally a polar body formed. When the surface force was calculated according to the cell shape, the surface force decreased at the animal pole but the force at the contractile ring increased. When by micromanipulation the mitotic apparatus was detached and translocated to the cortex other than the animal pole, polar body formation occurred all over the cortex of the oocyte, which indicates that the response of the whole cortex to the mitotic apparatus is equal. These results indicate that the decrease in the actin filaments and surface force near the centrosome of the mitotic apparatus as well as the increase in the actin filaments and surface force at some distance of the centrosome is important for cytokinesis.

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The widely held models of cytokinesis contend that signals for cleavage are transmitted by astral microtubules, and that such signals elicit the assembly and contraction of an equatorial band of actin-myosin II filaments. However, experiments during the past decade have painted an increasingly complex picture, including strong evidence for the involvement of chromosomal passenger proteins and interzonal microtubules, and the involvement of not only cortical contraction but also cytoskeletal disintegration. The purpose of this article is to consider alternative models that might better accommodate both old and new observations. It is proposed that chromosomal passenger proteins undergo dynamic associations at centromeres during metaphase and are recruited from the cytoplasm to both astral and interzonal microtubules during anaphase. In addition, cytokinesis may be driven by global inward contractions coupled to a localized collapse of the equatorial cortex.

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Phosphorylation of regulatory light chain (RMLC) of myosin II at Ser19/Thr18 is likely to play important roles in controlling the morphological changes seen during cell division of cultured mammalian cells. Phosphorylation of RMLC regulates the activity of myosin II, an essntial motor for cytokinesis, and phosphorylation of RMLC shows dramatic changes during mitosis. Two exzymes, myosin phosphatase and kinase, control phosphorylation of RMLC. Myosin phosphatase is activated during mitosis, apparently as a result of mitosis-specific phosphorylation of the myosin phosphatase targeting subunit (MYPT). This activation of myosin phosphatase is likely to result in RMLC dephosphorylation, causing the disassemly of stress fibers and focal adhesions during prophase. The phosphorylation of MYPT is lost in cyotokinesis, which would decrease myosin phosphatase activity. At the same time, ROCK (Rho-kinase) probably phosphorylates MYPT at its inhibitory sites, further decreasing the activity of myosin phosphatase. These changes in MYPT phosphorylation would raise RMLC phosphorylation, leading to the activation of myosin II for cyotokinesis. RMLC phosphorylation is also regulated by several RMLC kinases including ROCK (Rho-kinase), MLCK and citron kinase, all of which are localized at cleavage furrows. Future studies should examine whether these multiple kinases are redundant or whether they control distinct aspects of cell division.

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To identify the key molecules that regulate differentiation of hematopoietic cells, we carried out retrovirus-mediated functional screening for cDNAs whose expression suppresses IL-6-induced differentiation of mouse myeloid leukemic M1 cells. From this screening, we obtained a full length cDNA encoding a mouse homologue of human MgcRacGAP. Overexpression of the anti-sense MgcRacGAP profoundly inhibited IL-6-induced macrophage-differentiation of M1 cells. On the other hand, overexpression of the full-length form of MgcRacGAP alone enhanced macrophage differentiation of M1 cells in response to IL-6, and induced macrophage differentiation of HL-60 leukemic cells. To determine how this protein regulates differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical studies revealed that MgcRacGAP mainly localizes in the nucleus in interphase, accumulates on the mitotic spindle in metaphase, and is condensed in the midbody during cytokinesis. Overexpression of an N-terminal domain deletion mutant, which lacks the ability to localize to the midbody through association with tubulins, or a GAP-inactive mutant resulted in the formation of multinucleated cells in HeLa cells as well as in hemopoietic cells. Interestingly, MgcRacGAP in the midbody was phosphorylated probably on serine and threonine residues. These results indicate that MgcRacGAP regulates cytokinesis and cellular differentiation as a regulator of Rho family of GTPase and suggest that this process is controlled by some serine/threonine kinases.

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Chromosomal passenger proteins associate with chromosomes early in mitosis and transfer to the spindle at ana/telophase. Recent results show that aurora B/AIM-1 (aurora and Ipl1-like midbody-associated protein kinase), which is responsible for mitotic histone H3 phosphorylation, INCENP (Inner Centromere protein) and Survivin/BIR are in a macromolecular complex as novel chromosomal passenger proteins. Aurora B /AIM-1 can bind to Survivin and the C-terminal region of INCENP, respectively, and colocalizes with both proteins to the centromeres, midzone and midbody. Disruption of either aurora B/AIM-1 or INCENP function leads to sever defects in chromosome segregation and cytokinesis. Moreover, the formation of the central spindle through anaphase to cytokinesis is also disrupted severely.
These data suggest that chromosomal passenger complex is required for proper chromosome segregation by phosphorylating histone H3, and cytokinesis by ensuring the correct assembly of the midzone and midbody microtubule. Chromosomal passenger protein complex may couple chromosome segregation with cytokinesis.

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In the final stage of cell division, cytokinesis constricts and then seals the plasma membrane between the two daughter cells. The constriction is powered by a contractile ring of actin filaments, and scission involves rearrangement of the lipid bilayer of the cell membrane. We have shown that the lipid phosphatidylethanolamine (PE), which normally resides in the internal leaflet of the bilayer, is exposed on the external leaflet of the cleavage furrow as a result of enhanced transbilayer movement of the phospholipids during cytokinesis. To investigate the role of PE in cytokinesis, we employed two different approaches: manipulation of cell surface PE by a PE-binding peptide and establishment of a mutant cell line specifically defective in PE biosynthesis. Both approaches provide evidence that surface exposure of PE is essential for disassembly of the contractile ring at the final stage of cytokinesis. Based on these findings, we proposed that the transbilayer redistribution of PE plays a critical role in mediating coordinated movements between the contractile ring and the plasma membrane that are required for the proper progression of cytokinesis.

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Septins comprise a eukaryotic guanine nucleotide binding protein subfamily which form filamentous heteropolymer complexes. Although mechanism of cytokinesis is diverged by species and tissues, loss of septin function results in the multinuclear phenotype in many organisms. Hence septin filaments beneath the cleavage furrow are hypothesized as a structural basis to ensure completion of cytokinesis. However, molecular mechanisms of septin assembly, disassembly and function have been elusive despite the potential importance of this ubiquitous cytoskeletal system. Meanwhile, growing evidence suggests that mammalian septins functionally or physically interact with diverse molecules such as actin, actin-binding proteins, proteins of membrane fusion machinery, Cdc42 adapter proteins, a ubiquitin-protein ligase, and phosphoinositides. Careful integration of these data may provide insights into the mechanism of mammalian septin organization and functions in cytokinesis.

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Unconventional myosins in the budding yeast play essential roles in diverse cellular functions, inacluding endocytosis, actin organization, and polarized distribution of organelles. Several lines of evidence suggest that novel proteins, interacting with the unconventional myosins, regulate their functions. In this review, we focus on the functions of unconventional myosins from the point of view of myosin-interacting proteins.

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Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2^T18AS19A, substitution of both Ser19 and Thr18 by Ala; MRLC2^T18AS19D, Ser19 by Asp and Thr18 by Ala; and MRLC2^T18DS19D, both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2^T18DS19D contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2 ^T18AS19D. Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2^T18AS19A showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.

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The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.

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In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin monoclonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot analysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the α-helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an important component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs).

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We constructed stable mammalian cell lines in which human heterochromatin protein HP1α and kinetochore protein CENP-A were differentially expressed as fusions to red (RFP-HP1) and green fluorescent proteins (GFP-CENP-A). Heterochromatin localization of RFP-HP1 was clearly shown in mouse and Indian muntjac cells. By preparing mitotic chromosome spreads, the inner centromere localization of RFP-HP1 was observed in human and Indian muntjac cells. To characterize its molecular behavior in living mitotic cells, timelapse images of RFP-HP1 were obtained by computer-assisted image analyzing system, mainly with mouse cells. In G2 phase, a significant portion of RFP-HP1 diffused homogeneously in the nucleus and further dispersed into the cytoplasm soon after the nuclear membrane breakdown, while some remained in the centromeric region. Simultaneous observations with GFP-CENP-A in human cells showed that RFP-HP1 was located just between the sister kinetochores and then aligned to the spindle midzone. With the onset of anaphase, once it was released from there, it moved to the centromeres of segregating chromosomes or returned to the spindle equator. As cytokinesis proceeded, HP1α was predominantly found in the newly formed daughter nuclei and again displayed a heterochromatin-like distribution. These results suggested that, although the majority of HP1α diffuses into the cytoplasm, some populations are retained in the centromeric region and involved in the association and segregation of sister kinetochores during mitosis.

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