The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

Simulation testing of mechanisms for inducing the formation of the contractile ring in cytokinesis

There is persuasive evidence that the role of the mitotic apparatus (MA) in cytokinesis is to control the location of the cleavage furrow. The geometric aspects of this interaction between the MA and the cortex are complex and, thus, computer simulation can be a useful means for testing hypotheses about the induction process. White and Borisy (1983. J. Theor. Biol. 101:289-316) used computer simulations to show that long-range signals from the asters, varying inversely as various powers of distance, produce summed effects that are minima at the equator of spherical cells. Their results have seemed to support the "polar relaxation" class of hypotheses, in which the effect of the asters is to weaken cortical contractility so that contraction becomes maximized at the equator because it is least inhibited there. However, the experimental studies of Rappaport and Rappaport (1988. J. Exp. Zool. 247:92-98) indicate that the asters actually strengthen cortical contractility. In this paper, we use computer simulation to determine how signals from the MA will need to vary in effect as functions of distance to cause cortical contractility to become maximized where the furrows are to be induced. Although we confirm that inverse power inhibitory signals could induce equatorial furrows in spherical cells, we also find that this ability is destroyed by flattening, constricting, or distorting cells into cylinders, geometries for which Rappaport's experiments show furrows form (1986. Int. Rev. Cytol. 105:245-281). We then show that stimulatory signals of the right kind would induce furrows at the locations observed, in spherical cells as well as cells distorted by experimental manipulation. These signals must be constant out to a threshold distance but decrease abruptly beyond that distance. We also show that this ability depends on having the "drop-off" threshold occur at just the right distance relative to the dimensions of the cell and separation of the asters.     

Dissecting the role of disulfide bonds on the amyloid formation of insulin

Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenicity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6>INS-3>INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6-A11 caused a significant cytotoxicity increase and a higher potency to form high order toxic oligomers.     

Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.     

Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells.

Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 +/- 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.     

Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Gα13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Gα13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Gα13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.     

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.     

A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation

Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p.     

The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.     

Simultaneous inhibition of contractile ring and central spindle formation in mammalian cells treated with cytochalasin B

In this work we have used the inhibitor of F-actin polymerisation cytochalasin B (Cyt B) to test the hypothesis that the contractile ring and the central spindle are mutually interdependent structures in mammalian mitotic cells. Double fluorescence staining of α-tubulin and F-actin was employed to analyse anaphase and telophase figures from asynchronously growing cultures and prometaphase-synchronised cells. Testing for the presence of the central spindle and contractile ring in human primary fibroblasts, human hepatoma cells and Chinese hamster cells after Cyt B treatment showed that both structures were simultaneously absent in over 60% of treated anaphases and 80% of telophases. Experiments on resumption of cytokinesis in cleavage-arrested cells further showed that Cyt B-treated human fibroblasts proceeded to cleavage within minutes after removal of the drug from the medium, concomitant with the re-formation of both cellular structures in cleaving cells. These data suggest that the presence of a correctly assembled contractile ring is essential for the formation and persistence of the central spindle during ana-telophase and provide further support for the idea of a strong co-operative interaction between these two structures during cytokinesis. Received: 7 August 1998 / Accepted: 13 September 1998     

Timing of formation of Tetrahymena contractile ring microfilaments investigated by inhibition with skeletal muscle actin

Understanding the mechanism that determines the cell division plane is one of the most important problems in the fields of cell and developmental biology. Studying the timing and site of formation of contractile ring (CR) micro-filaments provides key information for solving the problem. We tried to create a nonfunctional CR in Tetrahymena by microinjecting rabbit skeletal muscle actin, which can copolymerize with Tetrahymena actin but has properties different from those of Tetrahymena actin. When skeletal muscle actin was injected in a predivision stage, before the onset of furrow constriction, long-term arrest of cell division was observed. Muscle actin did not cause any delay in cell division when the actin was injected at any stage other than the predivision stage. In all cases, muscle actin had little affect on other actin-related functions. Injected skeletal muscle actin polymerized near the equatorial division plane in cases of cell division arrest; it polymerized at other nonspecific locations when cell division was observed. Arrest occurred when the microinjection took place in the 17-min period just before the start of furrowing. This period coincides with the occurrence of equatorial deposits of p85, which is also suggested to be required for the determination of the division plane. The present experimental results are consistent with the idea that p85 is a crucial factor for determining the cell division plane and also functions as a polymerization nucleus for CR microfilaments. © 1992 Wiley-Liss, Inc.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Actin-depolymerizing protein Adf1 is required for formation and maintenance of the contractile ring during cytokinesis in fission yeast

The role of the actin-depolymerizing factor (ADF)/cofilin-family protein Adf1 in cytokinesis of fission yeast cells was studied. Adf1 was required for accumulation of actin at the division site by depolymerizing actin at the cell ends, assembly of the contractile ring through severing actin filaments, and maintenance of the contractile ring once formed. Genetic and cytological analyses suggested that it collaborates with profilin and capping protein in the mitotic reorganization of the actin cytoskeleton. Furthermore, it was unexpectedly found that Adf1 and myosin-II also collaborate in assembling the contractile ring. Tropomyosin was shown to antagonize the function of Adf1 in the contractile ring. We propose that formation and maintenance of the contractile ring are achieved by a balanced collaboration of these proteins.     

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.     

Dissecting the role of mTOR complexes in cellular senescence

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401. and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.     

Dissecting the role of mTOR complexes in cellular senescence

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401. and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.