Effects of anti-myosin drugs on anaphase chromosome movement and cytokinesis in crane-fly primary spermatocytes.

To investigate whether myosin is involved in crane-fly primary spermatocyte division, we studied the effects of myosin inhibitors on chromosome movement and on cytokinesis. With respect to chromosome movement, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) added during autosomal anaphase reversibly perturbed the movements of all autosomes: autosomes stopped, slowed, or moved backwards during treatment. BDM added before anaphase onset altered chromosome movement less than when BDM was added during anaphase: chromosome movements only rarely were stopped. They often were normal initially and then, if altered at all, were slowed. To confirm that the effects of BDM were due to myosin inhibition, we treated cells with ML-7, a drug that inhibits myosin light chain kinase (MLCK), an enzyme necessary to activate myosin. ML-7 affected anaphase movement only when added in early prometaphase: this treatment prevented chromosome attachment to the spindle. We treated cells with H-7 as a control for possible non-myosin effects of ML-7. H-7, which has a lower affinity than ML-7 for MLCK but a higher affinity than ML-7 for other potential targets, had no effect. These data confirm that the BDM effect is on myosin and indicate that the myosin used for chromosome movement is activated near the start of prometaphase. With respect to cytokinesis, BDM did not block furrow initiation but did block subsequent contraction of the contractile ring. When BDM was added after initiation of the furrow, the contractile ring either stalled or relaxed. ML-7 blocked contractile ring contraction when added at all stages after autosomal anaphase onset, including when added during cytokinesis. H-7 had no effect. These results confirm that the effects of BDM are on myosin and indicate that the myosin used for cytokinesis is activated starting from autosomal anaphase and continuing throughout cytokinesis.     

An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.     

Probing the role of septins in cardiomyocytes

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.     

The contractile ring

Summary Techniques of individual cell selection and precise ultramicrotomy have been employed to demonstrate that the contractile ring of cleaving HeLa cells is a transitory cytoplasmic organelle of distinctive fine structure and location. The contractile ring is an uninterrupted annulus encircling the equator of dividing cells exactly where the cleavage furrow forms. It is about 10 microns wide, up to 0.2 microns in thickness, and is composed exclusively of circumferentially aligned thin filaments 40–70 Å in diameter. Contractile ring filaments appear to be associated with the overlying plasma membrane.Controlled experiments with a mold metabolite (cytochalasin B) reveals that within a few minutes the drug abolishes the ability of HeLa cells to undergo cytokinesis. Cytochalasin B seems to decompose the contractile ring. It has no other clearly identifiable effects on other cell structures, notably the mitotic apparatus. Cytochalasin B is the only drug known which selectively inhibits cytokinesis in animal cells.In conclusion, the contractile ring is the most likely organelle responsible for cytokinesis in HeLa cells. Similar organelles probably occur in all cleaving animal cells. Successful cleavage depends upon the structural and functional integrity of the contractile ring.     

CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.     

Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast

A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2-4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7-9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum.     

Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow

Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.     

Mathematical Model of Contractile Ring-Driven Cytokinesis in a Three-Dimensional Domain

In this paper, a mathematical model of contractile ring-driven cytokinesis is presented by using both phase-field and immersed-boundary methods in a three-dimensional domain. It is one of the powerful hypotheses that cytokinesis happens driven by the contractile ring; however, there are only few mathematical models following the hypothesis, to the author’s knowledge. I consider a hybrid method to model the phenomenon. First, a cell membrane is represented by a zero-contour of a phase-field implicitly because of its topological change. Otherwise, immersed-boundary particles represent a contractile ring explicitly based on the author’s previous work. Here, the multi-component (or vector-valued) phase-field equation is considered to avoid the emerging of each cell membrane right after their divisions. Using a convex splitting scheme, the governing equation of the phase-field method has unique solvability. The numerical convergence of contractile ring to cell membrane is proved. Several numerical simulations are performed to validate the proposed model.     

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference-mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis.     

Mitochondria-cytoskeleton associations in mammalian cytokinesis

Background

The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.

Methods

We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.

Results

Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.

Conclusions

Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.

     

SulA-independent filamentation of<Emphasis Type="Italic"> Escherichia coli</Emphasis> during growth after release from high hydrostatic pressure treatment

To improve the efficiency of sterilization by high hydrostatic pressure treatment (HPT), it is desirable to know the biochemical process of bacteria most sensitive to the treatment. We investigated growth properties after release from HPT of exponentially growing Escherichia coli K-12 cells. We observed growth retardation after treatment (30 min at 37°C) above 75 MPa. Long filamentous cells of about eight times normal cell length were observed at 90 min growth after treatment at 75 MPa. In the subsequent period the filamentous cells divided into normal-sized cells. recA and sulA mutant strains also formed filamentous cells, indicating that filamentation was SulA-independent. Nucleoids segregated normally in the filamentous cells. Only one FtsZ ring (or none) was detected at possible division sites in the elongated cells. Western blotting analysis demonstrated that the amount of FtsZ protein was not affected by the treatment. GTP-dependent in vitro polymerization of either FtsZ protein in E. coli crude extract or purified FtsZ protein, however, was sensitive to HPT. These facts suggest that HPT at 75 MPa denatures a fraction of FtsZ molecules, and that these denatured molecules interfere with the polymerization of functional FtsZ, resulting in the significantly reduced number of FtsZ rings.     

Membrane lipid control of cytokinesis

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.     

A tropomyosin 1 induced defect in cytokinesis can be rescued by elevated expression of cofilin

Cytokinesis in eukaryotic cells is mediated by the contractile ring, an actomyosin-based structure which provides the force required to separate daughter cells. Isoforms of the actin-binding protein tropomyosin are also localised to the contractile ring in both fission yeast and human astrocytes. Although tropomyosin is required for cytokinesis in yeast, its precise role in the contractile ring is unknown. In this study we find that increased expression of a single tropomyosin isoform, tropomyosin 1, in U373MG astrocytoma cells leads to multinucleated cells and mitotic spindle defects. Furthermore, cells expressing increased levels of tropomyosin 1 usually fail to complete cytokinesis and this is accompanied by reduced accumulation of actin depolymerising factor/cofilin in the contractile ring. Adenovirus mediated expression of cofilin is able to relieve the tropomyosin 1 induced effects on cytokinesis. We conclude that tropomyosin 1 and cofilin play antagonistic roles within the contractile ring and that the balance between tropomyosin 1 and cofilin expression is important for cytokinesis.     

Verprolin cytokinesis function mediated by the Hof one trap domain

In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.     

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells

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.     

Temporal control of contractile ring assembly by Plo1 regulation of myosin II recruitment by Mid1/anillin

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes. These nodes then coalesce laterally into a functional contractile ring. Although Mid1 is present at the medial cortex throughout G2, recruitment of contractile ring components to nodes starts only at mitotic onset, indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis, and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset, coupling division plane specification to nuclear position. Here we provide evidence that Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2, and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset.     

Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.     

The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.     

Shs1 plays separable roles in septin organization and cytokinesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) form the septin ring at the bud neck during vegetative growth. We show here that disruption of SHS1 caused cold-sensitive growth in the W303 background, with cells arrested in chains, indicative of a cytokinesis defect. Surprisingly, the other four septins appeared to form an apparently normal septin ring in shs1Delta cells grown under the restrictive condition. We found that Myo1 and Iqg1, two components of the actomyosin contractile ring, and Cyk3, a component of the septum formation, were either delocalized or mislocalized in shs1Delta cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10Delta and cdc11Delta cells, respectively, suggesting that Shs1 is involved in septin organization, exerting different effects on septin-ring assembly, depending on the composition of the septin subunits. Furthermore, we constructed an shs1-100c allele that lacks the coding sequence for the C-terminal 32 amino acids. This allele still displayed the genetic interactions with the septin mutants, but did not show cytokinesis defects as described above, suggesting that the roles of Shs1 in septin organization and cytokinesis are separable.