The myriad roles of Anillin during cytokinesis

Anillin is a highly conserved multidomain protein that interacts with cytoskeletal components as well as their regulators. Throughout phylogeny, Anillins contribute to cytokinesis, the cell shape change that occurs at the end of meiosis and mitosis to separate a cell into daughter cells. Failed cytokinesis results in binucleation, which can lead to genomic instability. Study of Anillin in several model organisms has provided us with insight into how the cytoskeleton is coordinated to ensure that cytokinesis occurs with high fidelity. Here we review Anillin's interacting partners and the relevance of these interactions in vivo. We also discuss questions of how these interactions are coordinated, and finally provide some perspective regarding Anillin's role in cancer.     

Final stages of cytokinesis and midbody ring formation are controlled by BRUCE

Cytokinesis involves the formation of a cleavage furrow, followed by abscission, the cutting of the midbody channel, the final bridge between dividing cells. Recently, the midbody ring became known as central for abscission, but its regulation remains enigmatic. Here, we identify BRUCE, a 528 kDa multifunctional protein, which processes ubiquitin-conjugating activity, as a major regulator of abscission. During cytokinesis, BRUCE moves from the vesicular system to the midbody ring and serves as a platform for the membrane delivery machinery and mitotic regulators. Depletion of BRUCE in cell cultures causes defective abscission and cytokinesis-associated apoptosis, accompanied by a block of vesicular targeting and defective formation of the midbody and the midbody ring. Notably, ubiquitin relocalizes from midbody microtubules to the midbody ring during cytokinesis, and depletion of BRUCE disrupts this process. We propose that BRUCE coordinates multiple steps required for abscission and that ubiquitylation may be a crucial trigger.     

Talin concentrates to the midbody region during mammalian cell cytokinesis.

In this study we investigated the cellular distribution of talin, a cytoskeletal protein, during mammalian cell cytokinesis. Immunohistochemical experiments on various carcinoma cell lines and mesenchyme-derived cells reveal that talin displays a cell cycle-dependent cellular localization. During metaphase, talin is located in the centromeric region of the chromosome, like the TD-60 protein and intrinsic centromere components detected by a CREST serum. From anaphase to telophase, talin is present in the cleavage furrow. As the cells progress to cytokinesis, when the furrow is complete, talin is concentrated in the midbody structures, as assessed by immunofluorescence and confirmed by Western blot experiments on purified midbodies. Double staining experiments reveal that alpha-tubulin, TD-60 protein, and talin co-localize in the midbodies. These results suggest that talin, in addition to its implication in focal adhesion organization and signaling, may play a critical role in cytokinesis. (J Histochem Cytochem 47:1357-1367, 1999)     

Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors (reviewed in [1, 2]). Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA [3-7]. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. Here, we show that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis.     

The sesquiterpene lactone hymenoxon acts as a bifunctional alkylating agent

Hymenoxon, a toxic sesquiterpene lactone found in bitterweed, bound deoxyguanosine in a cell free system and formed adducts with guanine residues in cellular DNA. The reactive dialdehyde form of hymenoxon formed stable Schiff base products with deoxyguanosine which were separable from unreacted hymenoxon and deoxynucleosides by reverse phase high pressure liquid chromatography. Hymenoxon adducts which eluted as a single impure peak from the octadecylsilane column separated on amino and diphenyl-bonded phases with 10% methanol. Tritiated nucleoside adducts were isolated and purified from CFW mouse sarcoma cells treated with hymenoxon. Proton nuclear magnetic resonance spectra of purified hymenoxon-deoxyguanosine adducts revealed a loss of signals for hydroxyl groups in the bishemiacetal of hymenoxon. ¹³C-nuclear magnetic resonance spectra revealed that the major adduct has 35 carbon atoms, indicating an interaction of at least two guanine residues per hymenoxon molecule and suggesting that hymenoxon may cross-link DNA. Sedimentation analysis of treated DNA further showed that DNA cross-linking by hymenoxon (30 µg/ml) was equivalent to that of a known cross-linking agent, mitomycin C (7.5 µg/ml). Hymenoxon was more cytotoxic to DNA cross-link repair-deficient Chinese hamster ovary cell mutants than to repair proficient strains. These data combine to indicate that hymenoxon acts as a bifunctional alkylating agent which cross-links DNA in mammalian cells.CHO Chinese hamster ovary - HYM hymenoxon - MMC mitomycin C - NMR nuclear magnetic resonance - PBS phosphate buffered saline     

Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis

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Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

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.     

An anillin homologue,Mid2p,acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.     

Cyk3 acts in actomyosin ring independent cytokinesis by recruiting Inn1 to the yeast bud neck

Cytokinesis in yeast can be achieved by plasma membrane ingression, which is dependent on actomyosin ring constriction. Inn1 presumably couples these processes by interaction with both the plasma membrane and the temporary actomyosin ring component Hof1. In addition, an actomyosin ring independent cytokinesis pathway exists in yeast. We here identified Cyk3, a key component of the alternative pathway, as a novel interaction partner of Inn1. The carboxy-terminal proline rich part of Inn1 binds the SH3 domains of either Cyk3 or Hof1. Strains with truncated proteins lacking either of these SH3 domains do not display any severe phenotypes, but are synthetically lethal, demonstrating their crucial role in cytokinesis. Overexpression of CYK3 leads to an actomyosin ring independent recruitment of Inn1 to the bud neck, further supporting the significance of this interaction in vivo. Moreover, overexpression of CYK3 in a myo1 or an iqg1 deletion not only restores viability, but also the recruitment of Inn1 to the bud neck. We propose that Cyk3 is part of an actomyosin ring independent cytokinesis pathway, which acts as a rescue mechanism to recruit Inn1 to the bud neck.     

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.     

Rho1 directs formin-mediated actin ring assembly during budding yeast cytokinesis

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring.     

Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.     

Wound-induced contractile ring: a model for cytokinesis.

The actomyosin-based contractile ring is required for several biological processes, such as wound healing and cytokinesis of animal cells. Despite progress in defining the roles of this structure in both wound closure and cell division, we still do not fully understand how an actomyosin ring is spatially and temporally assembled, nor do we understand the molecular mechanism of its contraction. Recent results have demonstrated that microtubule-dependent local assembly of F-actin and myosin-II is present in wound closure and is similar to that in cytokinesis in animal cells. Furthermore, signalling factors such as small Rho GTPases have been shown to be involved in the regulation of actin dynamics during both processes. In this review we address recent findings in an attempt to better understand the dynamics of actomyosin contractile rings during wound healing as compared with the final step of animal cell division.     

Coordinating septum formation and the actomyosin ring during cytokinesis in Schizosaccharomyces pombe

During cytokinesis, animal and fungal cells form a membrane furrow via actomyosin ring constriction. Our understanding of actomyosin ring‐driven cytokinesis stems extensively from the fission yeast model system. However, unlike animal cells, actomyosin ring constriction occurs simultaneously with septum formation in fungi. While the formation of an actomyosin ring is essential for cytokinesis in fission yeast, proper furrow formation also requires septum deposition. The molecular mechanisms of spatiotemporal coordination of septum deposition with actomyosin ring constriction are poorly understood. Although the role of the actomyosin ring as a mechanical structure driving furrow formation is better understood, its role as a spatiotemporal landmark for septum deposition is not widely discussed. Here we review and discuss the recent advances describing how the actomyosin ring spatiotemporally regulates membrane traffic to promote septum‐driven cytokinesis in fission yeast. Finally, we explore emerging questions in cytokinesis, and discuss the role of extracellular matrix during cytokinesis in other organisms.     

Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis

Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.     

Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex

We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.