In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg

Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.     

Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP

Permeabilized PtK1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs. ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase does no require exogenous ATP and is unaffected by these inhibitors. These results are consistent with "dynein-like" ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.     

Microdissection of the mouse egg

A rapid and efficient method for microdissection of the mouse egg is described. The dissection is carried out in hanging drops of medium surrounded by heavy liquid paraffin oil at room temperature. Eggs are first deformed into a cylindrical shape and then dissected at a predetermined site with a glass needle on a Leitz micromanipulator. The survival rate of the dissected fragments is 75–90% and between 20 and 30 eggs can be dissected in an hour. Development of the dissected eggs is at least as good as that described after other types of manipulation. Cytoplasts and karyoplasts of various sizes can be prepared, as well as gynogenetic and androgenetic eggs with different amounts of cytoplasm. This procedure may help to examine nuclear-cytoplasmic interactions in eggs reconstituted from a variety of fragments.     

Meiosis: mouse eggs do their anaphase topsy-turvy

The meiotic separation of sister chromatids in mature metaphase II mouse eggs is observed to depend initially on spindle lengthening (Anaphase B), then?on microtubule shortening (Anaphase A). Having Anaphase B precede Anaphase A may be the mechanism by which mammalian eggs can generate a haploid chromosome number but without the loss of too much cytoplasm.     

Macrophage trafficking in the uterus and cervix precedes parturition in the mouse.

Immune activation is implicated in the etiology of preterm labor, but little is known about macrophage number or distribution in the uterus or cervix at term. This study tested the hypothesis that macrophages migrate into the reproductive tract before the onset of parturition. Paraffin-embedded sections from the mid-uterine horn and cervix of C3/HeN mice on Days 15 and 18 of pregnancy, the day of birth (Day 19), and 1 day postpartum were stained with a pan-macrophage marker to analyze cell numbers and distribution. During pregnancy, uterine macrophages were dispersed in endometrium, usually associated with vasculature and subluminal epithelium. In myometrium, macrophages were clustered in stromal connective tissue; near term and postpartum, cells appeared to surround the muscle bundles. Total macrophage numbers were increased on Day 15 relative to those in nonpregnant controls, declined before birth, and increased postpartum. In the cervix, macrophages congregated in subepithelium, often perivascular or near ganglia. Macrophage numbers in the cervix peaked on Day 18, then declined to nonpregnant levels by the day after birth. Thus, macrophage numbers in the uterus were inversely related to those in the cervix. These findings raise the possibility that macrophages and their products may be involved in uterine contractility and cervical remodeling during the processes of parturition.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

Cdc14-regulated midzone assembly controls anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle-regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase-Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase-Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase-Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase-Slk19 complex.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

A switch in microtubule dynamics at the onset of anaphase B in the mitotic spindle of Schizosaccharomyces pombe

Microtubule dynamics have key roles in mitotic spindle assembly and chromosome movement [1]. Fast turnover of spindle microtubules at metaphase and polewards flux of microtubules (polewards movement of the microtubule lattice with depolymerization at the poles) at both metaphase and anaphase have been observed in mammalian cells [2]. Imaging spindle dynamics in genetically tractable yeasts is now possible using green fluorescent protein (GFP)-tagging of tubulin and sites on chromosomes [3] [4] [5] [6] [7] [8]. We used photobleaching of GFP-labeled tubulin to observe microtubule dynamics in the fission yeast Schizosaccharomyces pombe. Photobleaching did not perturb progress through mitosis. Bleached marks made on the spindle during metaphase recovered their fluorescence rapidly, indicating fast microtubule turnover. Recovery was spatially non-uniform, but we found no evidence for polewards flux. Marks made during anaphase B did not recover fluorescence, and were observed to slide away from each other at the same rate as spindle elongation. Fast microtubule turnover at metaphase and a switch to stable microtubules at anaphase suggest the existence of a cell-cycle-regulated molecular switch that controls microtubule dynamics and that may be conserved in evolution. Unlike the situation for vertebrate spindles, microtubule depolymerization at poles and polewards flux may not occur in S. pombe mitosis. We conclude that GFP-tubulin photobleaching in conjunction with mutant cells should aid research on molecular mechanisms causing and regulating dynamics.     

Binding of low-density lipoproteins from chicken egg yolk to mouse anti-phospholipid B cells which include B cells against bromelain-treated mouse erythrocytes

Low-density lipoproteins from chicken egg yolk (EyLDL), which are reactive with mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC), were conjugated with fluorescein isothiocyanate (FITC). FITC-EyLDL could bind specifically to mouse anti-phospholipid B cells, which comprised all the BrMRBC-rosette-forming cells and anti-BrMRBC lipopolysaccharide-reactive B cells, C3H/He mice at 12 weeks of age had, approximately, 7 x 10(5) EyLDL-binding cells in the peritoneal cavity, 3 x 10(5) EyLDL-binding cells in the pleural cavity, and 3 x 10(5) EyLDL-binding cells in the spleen. In ontogeny, the numbers of EyLDL-binding cells in the peritoneal cavity expanded greatly by 4 weeks. Other normal strains of mice and C3H/HeJ mice at 12 weeks of age had 4-7 x 10(5) EyLDL-binding cells in the peritoneal cavity; the numbers were large (19 x 10(5] in NZB mice, rather small (2 x 10(5] in MRL/lpr mice, and very small (0.1 x 10(5] in CBA/N mice. In some of various strains of mice at 12 months of age, more than 20% of peritoneal cells were EyLDL-binding cells; in particular, all of five older NZB mice examined had more than 10(7) EyLDL-binding cells in the peritoneal cavity.     

Protein kinase C activity regulates the onset of anaphase I in mouse oocytes

The metaphase-to-anaphase I transition is a key step in the completion of meiosis I. In mouse oocytes, competence to exit metaphase I (MI) is developmentally regulated and typically not acquired until the preovulatory stage. The possible role of protein kinase C (PKC) in regulating this critical transition was assessed in both normal oocytes isolated from small antral follicles (18-day-old B6SJLF1 mice), which have not yet developed the capacity to progress to metaphase II (MII), and also oocytes defective in their ability to exit MI despite development to the preovulatory stage (24-day-old CX8 recombinant inbred strains). In both systems, transient suppression of endogenous PKC activity by treatment with a PKC-specific inhibitor, bisindolylmaleimide I (BIM), promoted the onset of anaphase I in a dose-dependent manner, while activation of PKC with the phorbol ester TPA blocked progression to MII. Following a 2-h incubation with BIM, the majority of oocytes progressed to, and arrested at, MII. The resulting MII oocytes were fertilizable in vitro, showing similar cleavage and blastocyst development rates between BIM treated and untreated controls. Transferred embryos resulted in the development of pups to term in both groups. These data demonstrate that PKC plays an important role in regulating the onset of anaphase I in mouse oocytes. Moreover, it is concluded that oocytes isolated from small antral follicles become blocked at MI due to a PKC-mediated signal, suggesting that acquisition of competence to complete meiosis I involves, in part, the control of PKC activity. Similarly, failure to regulate PKC activity at the preovulatory stage likely promotes arrest at MI.     

Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics

We established a conditional deletion of Aurora A kinase (AurA) in Cdk1 analogue-sensitive DT40 cells to analyze AurA knockout phenotypes after Cdk1 activation. In the absence of AurA, cells form bipolar spindles but fail to properly align their chromosomes and exit mitosis with segregation errors. The resulting daughter cells exhibit a variety of phenotypes and are highly aneuploid. Aurora B kinase (AurB)-inhibited cells show a similar chromosome alignment problem and cytokinesis defects, resulting in binucleate daughter cells. Conversely, cells lacking AurA and AurB activity exit mitosis without anaphase, forming polyploid daughter cells with a single nucleus. Strikingly, inhibition of both AurA and AurB results in a failure to depolymerize spindle microtubules (MTs) in anaphase after Cdk1 inactivation. These results suggest an essential combined function of AurA and AurB in chromosome segregation and anaphase MT dynamics.     

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.     

Metabolic inhibitors block anaphase A in vivo

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to freeze in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.     

Sperm tail entry into the mouse egg in vitro

Cumulus-free mouse eggs were placed on microscope slides and inseminated with capacitated mouse spermatozoa. Fertilization could then be observed through the phase contrast microscope and recorded by time-lapse cinematography. Following the penetration of the fertilizing spermatozoon through the zona pellucida and the fusion of the sperm head with the vitelline membrane, the entire sperm tail gradually entered the vitellus. The time required for tail incorporation into the vitellus as measured in 49 eggs varied from 3 h 3 min to 5 h 49 min, with a mean time of 4 h 23 min. When tail incorporation began, the greater part of the flagellum was still outside the zona pellucida; occasionally it slipped into the perivitelline space, but generally it remained outside the zona and shortened by degrees as incorporation proceeded. The motility of the fertilizing spermatozoon declined abruptly very soon after fusion of the sperm head with the vitellus and remained at a very low level during the 3–6 h required for tail incorporation. Sperm motility, therefore, does not appear to be the main determinant in tail incorporation and the primary mechanism responsible for it remains unclear. As the sperm tail slowly entered the vitellus, the second meiotic division was completed with concomitant extrusion of the second polar body. Key stages in second polar body formation were correlated with events in tail incorporation. Differences between fertilization in vitro and in vivo are discussed.     

Oscillatory CaMKII activity in mouse egg activation

Fertilization-induced intracellular calcium (Ca(2+)) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca(2+) signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca(2+) similar to those at fertilization stimulate oscillatory Ca(2+)/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca(2+) signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca(2+) levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in Ca(2+). Enzyme activity increased 370% during the first Ca(2+) rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca(2+) reached its peak value. Single fertilized eggs monitored for Ca(2+) had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca(2+) transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca(2+) and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca(2+) in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation.