Interdependence of the contractile ring and spindle midzone in cleavage plane maintenance

How segregation of the chromosomes is coordinated with the ensuing cell cleavage to complete the cell cycle is not well understood. A recent study of cytokinesis in fission yeast by Pardo and Nurse suggests that the contractile ring is required for assembly of the post-mitotic microtubule array (PAA). In turn, the PAA is required to maintain the contractile ring at the cleavage plane, as well as to keep the nuclei separated at the poles of the cleaving cell. These functions may be particularly important for a cell cycle checkpoint ensuring that if cytokinesis is delayed, septation will occur between the two daughter nuclei.     

Cell division of binuclear cells induced by caffeine: Spindle organization and determination of division plane

Synchronously dividing binuclear cells were induced in root tips ofTriticum turgidum by caffeine treatment. Spindle and other microtubular configurations of such cells were studied using tubulin immunofluorescence and electron microscopy. The binuclear cells developed one, two or three preprophase microtubule bands longitudinally, transversely or rarely in a cross configuration. During the mitotic entry binuclear cells formed prophase spindles separately around each nucleus. When the nuclei were located fairly apart, their spindle structures developed independently throughout all mitotic phases. But when the nuclei were located closely together their metaphase and anaphase spindles shared a common polar region. However, the two spindles in such cells retained their functional autonomy. They display structurally independent minipoles in the common polar region. After anaphase the neighbouring nonsister chromosome groups of nuclei divided by a common polar region come to lie close together and in telophase, become enclosed by a common nuclear envelope. During cytokinesis of binuclear cells cell plates were formed only between sister nuclei. These cell plates may develop normally or may curve or branch giving rise to aberrant daughter cell walls. The peculiar mode of spindle and spindle polar region organization of binuclear cells and determination of the division plane in them are discussed.     

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.     

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.     

Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells

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.     

RNA synthesis and cell division in cold-synchronized cells of Tetrahymena pyriformis

Cells of Tetrahymena pyriformis have been cold-synchronized using a repetitive cycle of six, two-hour cold shocks (9.5°C) alternating with decreasing periods (60–30 minutes) at 28°C. This system gives a maximum division index of 70–80% occurring at 90 minutes from the end of the last synchronizing cold-treatment (EC). Examination of the division sensitivity of these cells to actinomycin D applied continuously at ten-minute intervals from EC reveals that division is essentially blocked until approximately 40 minutes past EC, after which a rapid decrease is sensitivity to the inhibitor occurs. Coinciding with this period of high sensitivity is the occurrence of a peak of C¹⁴ uridine incorporation at 40 minutes past EC. Inhibition of this peak is correlated with an inhibition of division, whereas strong inhibition of RNA synthesis beyond 60 minutes past EC has little effect on division activity. The similarity of these findings with those of the heat-synchronized system is discussed with the suggestion that both heat- and cold-synchronizing treatments result in the synchronous resynthesis of a division-associated fraction of RNA.     

Tetrahymena profilin is localized in the division furrow.

Localization of Tetrahymena profilin was examined by an immunofluorescence method. In interphase Tetrahymena cells, immunofluorescence for profilin was diffusely distributed in the cytoplasm, while in dividing cells, additional intense fluorescence was observed in the division furrow. From the result of immunofluorescence localization using cytoskeletal cell models, a significant fraction of profilin appeared to become insoluble in association with a cytoskeletal structure just beneath the division furrow during cytokinesis, although remaining profilin existed as a soluble form in the cytoplasm. Double immunofluorescence staining with anti-profilin and anti-actin antibodies revealed that the localization of profilin in the division furrow coincided with that of contractile ring microfilaments in terms of both position and timing. This is the first report describing the coexistence of profilin with actin filaments in the division furrow, implying the possible involvement of profilin in assembly and disassembly of contractile ring microfilaments in the process of cytokinesis.     

Mechanisms of contractile ring tension production and constriction

The contractile ring is a remarkable tension-generating cellular machine that constricts and divides cells into two during cytokinesis, the final stage of the cell cycle. Since the ring’s discovery, the parallels with muscle have been emphasized. Both are contractile actomyosin machineries, and long ago, a muscle-like sliding filament mechanism was proposed for the ring. This review focuses on the mechanisms that generate ring tension and constrict contractile rings. The emphasis is on fission yeast, whose contractile ring is sufficiently well characterized that realistic mathematical models are feasible, and possible lessons from fission yeast that may apply to animal cells are discussed. Recent discoveries relevant to the organization in fission yeast rings suggest a stochastic steady-state version of the classic sliding filament mechanism for tension. The importance of different modes of anchoring for tension production and for organizational stability of constricting rings is discussed. Possible mechanisms are discussed that set the constriction rate and enable the contractile ring to meet the technical challenge of maintaining structural integrity and tension-generating capacity while continuously disassembling throughout constriction.