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.     

Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.
The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.
The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.     

Unilateral and wandering furrows during mitosis in vertebrates: implications for the mechanism of cytokinesis

Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction and wander for >40 microm through the cell. These observations reveal that the 'contractile ring' normally seen during cytokinesis is composed of multiple independent 'furrowing units' that are normally coordinated to form a symmetrical furrow around the cell, and that once formed this so-called contractile band does not function as a 'purse string' as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this propagation occurs remains an important question for the future.     

Effects of the phosphatase inhibitors,okadaic acid,ATPgammaS, and calyculin A on the dividing sand dollar egg

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.     

Taxol-stabilized microtubules can position the cytokinetic furrow in mammalian cells

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.     

On the mechanism of cleavage furrow ingression in Dictyostelium

The ability of Dictyostelium cells to divide without myosin II in a cell cycle-coupled manner has opened two questions about the mechanism of cleavage furrow ingression. First, are there other possible functions for myosin II in this process except for generating contraction of the furrow by a sliding filament mechanism? Second, what could be an alternative mechanical basis for the furrowing? Using aberrant changes of the cell shape and anomalous localization of the actin-binding protein cortexillin I during asymmetric cytokinesis in myosin II-deficient cells as clues, it is proposed that myosin II filaments act as a mechanical lens in cytokinesis. The mechanical lens serves to focus the forces that induce the furrowing to the center of the midzone, a cortical region where cortexillins are enriched in dividing cells. Additionally, continual disassembly of a filamentous actin meshwork at the midzone is a prerequisite for normal ingression of the cleavage furrow and a successful cytokinesis. If this process is interrupted, as it occurs in cells that lack cortexillins, an overassembly of filamentous actin at the midzone obstructs the normal cleavage. Disassembly of the crosslinked actin network can generate entropic contractile forces in the cortex, and may be considered as an alternative mechanism for driving ingression of the cleavage furrow. Instead of invoking different types of cytokinesis that operate under attached and unattached conditions in Dictyostelium, it is anticipated that these cells use a universal multifaceted mechanism to divide, which is only moderately sensitive to elimination of its constituent mechanical processes.     

Transitions regulating the timing of cytokinesis in embryonic cells

Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.     

Direct, high-resolution measurement of furrow stiffening during division of adherent cells

It is unclear whether cell division is driven by cortical relaxation outside the equatorial region or cortical contractility within the developing furrow alone. To approach this question, a technique is required that can monitor spatially-resolved changes in cortical stiffness with good time resolution. We employed atomic force microscopy (AFM), in force-mapping mode, to track dynamic changes in the stiffness of the cortex of adherent cultured cells along a single scan-line during M phase, from metaphase to cytokinesis. Video microscopy, which we used to correlate the AFM data with mitotic events identified by light microscopy, indicated that the AFM force-mapping technique does not perturb dividing cells. Here we show that cortical stiffening occurs over the equatorial region about 160 seconds before any furrow appears, and that this stiffening markedly increases as the furrow starts. By contrast, polar relaxation of cells does not seem to be an obligatory event for cell division to occur.     

Microtubules are required for completion of cytokinesis in sea urchin eggs.

Completion of cytokinesis, abscission, has been studied little despite the intensive studies of the onset and contractile mechanism of the earlier phases of division. It has been well documented that microtubule (MT) disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed. We have confirmed those findings using the MT inhibitors, nocodazole and demecolcine. In addition, we have found that MT disruption after furrow stimulation but before completion of division prevents abscission as evidenced by the observation that prospective daughter cells in MT-disrupted eggs maintain electrical continuity. Continued observation of eggs revealed that the furrow in MT-disrupted eggs did not result in abscission, but rather held steady until the time when controls underwent second cleavage, at which point the furrows regressed. These findings extend the recent reports that MTs are required for completion of division in mammalian tissue culture cells and frog eggs, to invertebrates, suggesting a common mechanism of abscission for animal cells.     

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.     

Simulation of density gradients of astral microtubules at cell surface in cytokinesis of sea urchin eggs

Astral microtubules extend close to the cell surface just before cytokinesis in sea urchin eggs. At this time, a small region with a constant area is considered around a point on the egg surface. To calculate the number of microtubules that reach the surface region, i.e. the microtubule density at the point, a simple mathematical model was set up. The density was estimated at many surface points in multipolar and distorted eggs by using the model. A contour map was drawn to investigate the density gradients. The gradient patterns were compared with the distributions of contractile-ring microfilaments. The simulated cases were: (1) an unusual distribution of contractile-ring microfilaments in an egg that had polyasters and was compressed by a coverslip; (2) formation of contractile-ring microfilaments at the equatorial region in compressed eggs with a centrally-located mitotic apparatus; (3) normal furrowing in the plane of the spindle midpoint in eggs inserted into a glass loop or confined in a capillary; (4) failure of furrow formation in spherical eggs treated with ethyl urethane and revival of furrowing by pushing the equatorial surface close to the spindle. These simulations proposed the hypothesis that contractile-ring microfilaments form at surface regions where the microtubule density has a local minimum, not a local maximum. In addition, it was suggested that the probability of the formation of the contractile-ring microfilaments is dependent on how abruptly the density gradient changes at the local-minimum point. These results support the idea that the gradient pattern of the microtubule density determines whether and where contractile-ring microfilaments appear.     

Determination of the First Two Cleavage Furrows in Developing Eggs of Triturus Alpestris Compared with Other Forms

Eggs of Triturus alpestris were horizontally compressed at times before first and second cleavage in an experiment designed to measure the time at which the mitotic apparatus determines the direction of the subsequent cleavage furrow. The results showed that furrow determination was completed 0.46 Dettlaff units before the onset of furrowing. When a comparison was made of the times of furrow determination before cleavage in eggs of different animal groups using Dettlaff units, the results supported the idea that preparations for furrowing proceed differently in echinoderm eggs from eggs of sturgeon and amphibia.     

Furrow Constriction in Animal Cell Cytokinesis

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.     

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.     

The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.     

Furrow Constriction in Animal Cell Cytokinesis

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.     

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

The BTB protein MEL-26 promotes cytokinesis in C. elegans by a CUL-3-independent mechanism

BACKGROUND: The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS: Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS: Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.     

Inducing “cytokinesis” without mitosis in unfertilized Drosophila eggs

Selection of the cleavage plane during cytokinesis in dividing cells is linked to the position of the mitotic spindle. A major player in cleavage plane positioning is believed to be the anaphase central spindle and its associated signaling complex called centralspindlin, composed of MgcRacGap and MKLP1. Centralspindlin has the capacity to induce furrowing of the cell cortex by promoting the localized activation of RhoA, which in turn promotes assembly of the contractile ring. We have found a way to induce a cytokinesis-like process in unfertilized Drosophila eggs and very early embryos, when spindle structures are few and located far from invaginating egg cortex. The simple injection of a small molecule inhibitor of Cdk1/Cyclin B (either Roscovitin or RO3306) is sufficient to promote membrane invagination near the site of injection. The furrow generated is in many respects similar to a classical cleavage furrow. Actin, myosin, anillin and MKLP1 are all associated with the forming furrow, which in some cases can entirely circumscribe the unfertilized egg. A similar furrow can also be generated by the localized injection of constitutively active RhoA protein, suggesting that Cdk1 is normally an upstream inhibitor of RhoA activation. We show further that this process apparently is not associated with microtubules. Since simple localized inhibition of Cdk1 is sufficient to induce a furrow, we suggest that in real cytokinesis in normal cells, the localized downregulation of Cdk1 activity at the metaphase-anaphase transition may contribute, along with the spindle, to the positioning of the cleavage furrow.     

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution.
The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10⁻³ dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10⁻³ dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10⁻³ dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.