Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.     

Mitotic apparatus-surface interaction and cell division

Summary

Results of recent investigations concerning the mechanisms of animal cell division are reviewed. The mitotic apparatus was aspirated from a blastomere of a sand dollar (Echinarachnius parma) egg before second cleavage, and the time interval between removal and the appearance of the furrow in the control companion blastomere was measured. When the mitotic apparatus is removed 4 min or less before the furrows appear in the controls, furrows also develop in the operated cells. These results show that 4 min before furrowing begins, the surface changes which lead to formation of the division mechanism have become irreversible. When the mitotic apparatus of a cylindrical cell is shifted by pushing in one of the poles when the furrow appears, a new furrow develops in association with the new position of the mitotic apparatus. The same mitotic apparatus could elicit as many as 13 furrows over a 24.5 min period following the appearance of the first furrow. The results show that, in the proper geometrical circumstances, the mitotic apparatus and the surface can interact over a longer period than they do in normal cells.

By artificially constricting sand dollar eggs with a glass loop, the normal distance relations between the astral centers and the polar and equatorial surfaces can be reversed. Constricted cells cleave normally. The blocking effect of ethyl urethane can be reversed by moving the equatorial surface closer to the spindle portion of the mitotic apparatus. Relocation of other parts of the surface closer to the mitotic apparatus was ineffective. These results help elucidate the geometrical relations that are essential for furrow formation between the mitotic apparatus and the surface.

In cylindrical sand dollar eggs, single asters and the widely separated asters of a broken mitotic apparatus can cause furrow-like constrictions in the adjacent cylindrical surface. This reaction can be blocked by treating cells with ethyl urethane, which reduces astral size. The nature of the shape change that the aster causes depends upon the surface region affected. These results aid in understanding the nature of the change in surface physical activity caused by the mitotic apparatus.     

Chromosomal Proteins and Cytokinesis: Patterns of Cleavage Furrow Formation and Inner Centromere Protein Positioning in Mitotic Heterokaryons and Mid-anaphase Cells

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Action at a distance during cytokinesis

Animal cells decide where to build the cytokinetic apparatus by sensing the position of the mitotic spindle. Reflecting a long-standing presumption that a furrow-inducing stimulus travels from spindle to cortex via microtubules, debate continues about which microtubules, and in what geometry, are essential for accurate cytokinesis. We used live imaging in urchin and frog embryos to evaluate the relationship between microtubule organization and cytokinetic furrow position. In normal cells, the cytokinetic apparatus forms in a region of lower cortical microtubule density. Remarkably, cells depleted of astral microtubules conduct accurate, complete cytokinesis. Conversely, in anucleate cells, asters alone can support furrow induction without a spindle, but only when sufficiently separated. Ablation of a single centrosome displaces furrows away from the remaining centrosome; ablation of both centrosomes causes broad, inefficient furrowing. We conclude that the asters confer accuracy and precision to a primary furrow-inducing signal that can reach the cell surface from the spindle without transport on microtubules.     

Cell cleavage : Ultrastructural evidence against equatorial stimulation by aster microtubules

Cell cleavage is spatially and temporally coordinated with karyokinesis. In astral division, as occurs in sea urchin eggs, coordination is accomplished by the mitotic asters. We have explored the following hypotheses:

1. 1. That microtubules of the two asters cross at the cell's equator.

2. 2. That because they cross, or by some other configuration, more microtubules interact with the equatorial cortex than with the polar cortex.

3. 3. That the microtubule component of astral rays differentially stimulates the equatorial cortex for cleavage contraction.

Using a fixation procedure which enhances visibility of microtubules, we have determined that aster microtubules do not cross at the equatorial cortex at any stage of mitosis relevant to cleavage stimulation, contrary to the first hypothesis. Aster microtubules extend progressively farther during anaphase, but the two arrays occupy mutually exclusive hemispheres in the egg. Using another fixation procedure which results in more conventional microtubule morphology, we have systematically counted microtubules penetrating the cortex at both the equator and the poles in sections cut parallel and perpendicular to the axis of the mitotic apparatus, respectively, at all stages of mitosis. We did not observe any microtubules in the cortex of the equator during prometaphase, metaphase, early anaphase or mid-anaphase. In comparison, small numbers of microtubules were observed in the polar cortex during this time. By late anaphase there are some microtubules in the equatorial cortex but many more are observed in the polar cortex. These findings are contrary to the second hypothesis and therefore do not establish the morphological basis for the third hypothesis. We conclude that there is no positive correlation between microtubule numbers at the egg equator and the timing of cleavage stimulation. Therefore, coordination between karyokinesis and cell cleavage is achieved by some process other than the simple numerical increase of microtubules at the equatorial cortex.     

Division of constricted and urethane-treated sand dollar eggs: a test of the polar stimulation hypothesis

In spherical cells with a central mitotic apparatus, the centers of the asters are closer to the poles than to the equator. This circumstance is basic to several hypothetical explanations of the way in which the mitotic apparatus establishes the division mechanism. This investigation was designed to determine whether that geometrical relationship is necessary for division. Fertilized, mechanically denuded sand dollar eggs were inserted into glass loops, which reduced the diameter in the constriction plane from the normal 142 to 78-80 microns and partly constricted the cell into equal parts. The mitotic apparatus straddled the constriction, and its length was not significantly changed. The manipulation increased the distance from the astral centers to the poles and decreased the distance from the astral centers to the equator to a degree that reversed the normal distance relations. These cells divided normally. Ethyl urethane (0.06 M) reduces the size of the mitotic apparatus and blocks cleavage in spherical cells. When treated cells are confined in 80-microns i.d. capillaries, they divide. Treated cells also divide when they are constricted by an 80-microns i.d. glass loop if the mitotic apparatus straddles the constriction. An equal degree of constriction in the subfurrow and subpolar areas did not reverse the effect of urethane. The results demonstrate that cleavage does not depend on the normal distance relation between the mitotic apparatus and the poles, and that the urethane effect can be remedied only by reducing the distance between the mitotic apparatus and the equatorial surface. Both findings are inconsistent with the polar stimulation hypothesis.     

Multipolar mitosis in procaine-treated polyspermic sea urchin eggs and in eggs fertilized with UV-irradiated spermatozoa with a computer model to simulate the positioning of centrosomes

Procaine-treated eggs can be penetrated by more than one spermatozoon. Supernumerary male pronuclei can fuse with the female one giving raise to multipolar spindles or remain isolated within the egg's cytoplasm forming their own spindle. In all types of multiple mitotic figures (asters and spindles) the distribution of asters is equidistant either uniplanar or at maximum distance like at the apices of a polyhedron. Astral rays are not different from spindle fibers: they can attach to and attract chromosomes of "foreign" mitotic figures. When several mitotic figures are present in one egg, the partner asters are always of the same size, and microtubules of one aster never interdigitate with those of others. The hypothesis that positioning of centrosomes is brought about by spreading of a centrosome organizer in the form of an expanding calotte on the surface of the nucleus (Mazia, D., Int. Rev. Cytol. 100, 49-92 (1987)) is supported by a computer model.     

Intracellular distribution of calcium and phosphorus during the first cell division of the sea urchin egg

The intracellular distribution of calcium and phosphorus during metaphase and anaphase of the first cleavage in sea urchin eggs was studied with the electron-probe microanalyzer. This study allowed a comparison of the relative concentrations of both elements on the polar and cleavage furrow regions of the membrane and on the mitotic asters and cytoplasm. The results show that in most eggs, both calcium and phosphorus are more highly concentrated in the mitotic asters than in surrounding cytoplasm during both anaphase and metaphase. Calcium is more concentrated at the furrow region than at the polar region during metaphase but not anaphase. The role of calcium during mitosis was reviewed with special reference to the theories on the formation of the cleavage furrow along the equatorial zone between two mitotic centers.     

The astral relaxation theory of cytokinesis revisited

Cytokinesis in animal cells is accomplished by the active constriction of the equatorial regions of a cell by an actomyosin-containing contractile ring. The mitotic apparatus specifies the position and orientation of the furrow such that the mitotic spindle is always bisected. Global cortical contractions occur in the cortex of a cell prior to cytokinesis that are independent of the presence of the mitotic apparatus. It was proposed some years ago that the asters of the mitotic apparatus could act to relax the preformed cortical tension in their vicinity. This would produce a differential in tension between the equatorial regions and the adjacent regions of the cortex so that the equatorial regions would contract, forming a cleavage furrow. It can be shown that, as it stands, this theory cannot explain cleavage. However, if cortical contractile elements are assumed to be laterally mobile in the plane of the cortex, then the astral relaxation theory can account for many of the aspects of cleavage, including the formation of the contractile ring. Similar schemes may account for the behaviour of the lamellapodia of motile cells.     

Two-step positioning of a cleavage furrow by cortexillin and myosin II

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.     

Induction of cytokinesis is independent of precisely regulated microtubule dynamics

Astral microtubules (MTs) emanating from the mitotic apparatus (MA) during anaphase are required for stimulation of cytokinesis in eggs. We have used green fluorescent protein-labeled EB1 to observe MT dynamics during mitosis and cytokinesis in normal sea urchin eggs. Analysis of astral MT growth rates during anaphase shows that MTs contact the polar cortex earlier than the equatorial cortex after anaphase onset but that a normal cleavage furrow is not induced until contact with MTs has been achieved throughout the cortex. To assess the role of MT dynamics in initiation of cytokinesis, we used a collection of small molecule drugs to affect dynamics. Hexylene glycol resulted in rapid astral elongation due to decreased MT catastrophe and precocious furrowing. Taxol suppressed MT dynamics but did not inhibit furrow induction when the MA was manipulated toward the cortex. Urethane resulted in short, highly dynamic astral MTs with increased catastrophe that also stimulated furrowing upon being brought into proximity to the cortex. Our findings indicate that astral MT contact with the cortex is necessary for furrow initiation but that the dynamic state of astral MTs does not affect their competency to stimulate furrowing.     

On the mechanisms of cytokinesis in animal cells

We present a model that attempts to explain some aspects of cytokinesis in animal cells. We propose two separate phases of cytokinesis. The first is not dependent on the presence of the mitotic apparatus and involves a general activation of cortical contractile elements resulting in the development of a surface tension. In the second phase the asters of the mitotic apparatus interact and modulate the activities of the tension generating elements in the cortex to produce gradients of surface tension with the highest values being at the equator. Tension generating elements are assumed to be free to move in the plane of the cortex so that they will consequently move up the gradient of tension and accumulate as an equatorial belt of oriented elements i.e. the contractile ring. The model was simulated on a computer and is capable of reproducing some of the wide variety of cleavage configurations that are observed.     

Anillin promotes astral microtubule-directed cortical myosin polarization

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle-directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

Cleavage Furrow Establishment--A Preliminary to Cylindrical Shape Change

The predictable pattern of cell shape changes characterizinganimal development must be a consequence of control mechanismsthat are at least analogous to those operating in dividing cells.When cells change shape by an internal mechanism, it is impliedthat they also contain systems that will activate and deactivatethe mechanism, localize it within the cell, insure proper timing,and impart the proper vectoral qualities. Experimental investigationsdesigned to elucidate similar processes in dividing cells revealthat the physical mechanism that accomplishes cytokinesis isestablished at or near the equatorial cell surface by the mitoticapparatus. The process is completed by late metaphase or earlyanaphase. In cleaving eggs a pair of asters can substitute forthe intact mitotic apparatus. The nature of the stimulus whichapparently passes from the mitotic apparatus to the surfaceis presently unknown. It moves toward the surface at about 6microns per minute and requires about 1 minute to establishthe mechanism. The resulting equatorial contractile activityis initially isotropic but becomes anisolropic at the beginningof visible constriction.     

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.     

A Highlights from MBoC Selection: Analyzing the Effects of Delaying Aster Separation on Furrow Formation during Cytokinesis in the Caenorhabditis elegans Embryo

Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow. Here, we explore the contribution of the asters by analyzing the consequences of altering interaster distance during the first cytokinesis of the Caenorhabditis elegans embryo. Delaying aster separation, by using TPXL-1 depletion to shorten the metaphase spindle, leads to a corresponding delay in furrow formation, but results in a single furrow that ingresses at a normal rate. Preventing aster separation, by simultaneously inhibiting TPXL-1 and Gα signaling-based cortical forces pulling on the asters, delays furrow formation and leads to the formation of multiple furrows that ingress toward the midzone. Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold. Simultaneously delaying aster separation and disrupting midzone-based signaling leads to complete failure of furrow formation. Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

Adhesion-dependent and contractile ring-independent equatorial furrowing during cytokinesis in mammalian cells

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 microM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 microM), which inhibits Rho-kinase, was similar to 30 microM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 microM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 microM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.