Action de l'hydrate de chloral sur les mitoses de segmentation de l'œuf de pleurodèle

In eggs of Pleurodeles treated with chloralhydrate (0.1 M) spindle and astral fibers are progressively destroyed after 4 hours, leading to apolar nuclei, apolar mitoses and “monopolar” mitoses, the so-called star metaphases. After 1 hour the spindle is shortened, but not narrowed and separated from the poles and asters. Its microtubules, grown before metaphase, are first inhibited at their ends near the centrospheres. After 4 hours, defibrillated achromatic material, stained by methyl blue, surrounds a clearer zone originating from the nucleoplasm, in which chromosomes are embedded. At the EM level the treatment induces the formation of unusual tubular bodies connected with the centrospheres and of similar bodies related to kinetochores and chromosomes. These bodies are formed of tubular residues, parallel or in concentric systems, the latter embedded in a matrix containing tightly packed filaments of 170 Å diameter. The star metaphase is characterized by homogeneous centrospheres formed only of filaments and completely independent from kinetochores and chromosomes. Chromosomes are radially distributed around a central common mass, which keeps the chromosomes together; it is formed of a finely fibrous matrix containing disordered microtubular residues; kinetochores are embedded in the common mass. Fuzziness and alteration of chromosomes proceed as a direct action of the chloralhydrate. The star metaphase is not a real “monopolar” mitosis.     

L'égalité fonctionnelle des pôles dans les mitoses pluripolaires déterminées par le phényluréthane

The multiple poles formed under the influence of phenylurethane are nearly equal in attraction power and fibrillogenetic activity: the centrospheres are therefore of equal volume. There are no principal or secondary poles, as in the action of selenium dioxide. The “block of centrospheres” by quinoline derivatives was used to make evident these conclusions: 7 to 11 blocked centrospheres appeared around the chromosomes, or at prophase, but never at telophase. In other cases addition of the antimitotic actions results in a lessening of polar activity. The multiplication of equally active cellular centers is demonstrated in this way. Unpaired numbers of blocked centrospheres and different numbers of the two groups of centrospheres formed are consequences of asynchronism determined by lenghtening of the end phases of mitoses and inhibition of fibrillogenesis. The disposition of the centrospheres on two perpendicular axes is determined by the conditions of centriolar reproduction.     

Action des acides caproïque et caprylique sur les mitoses de segmentation chez Triturus helveticus Razoumowsky

Caproic (hexanoic) and caprylic (octanoic) acids 0,01 M (pH 5) are comparatively studied. After 3 hours they do not destroy spindle fibers, but produce several abnormalities classified and hierarchized as follows: 1) bipolar mitoses with inactive poles and blocked centrospheres, 2) mitoses with two dominant poles including centrospheres and secondary poles without them, 3) special pluripolar mitoses with an equatorially dissociated spindle and more or less efficient poles. Partial inhibition of polar activity results in extended hyperfibrillar diastema, which proliferates in fibrillar hollow spheres and burses, but is unable of inducing furrowing. Mechanisms of these disturbances are discussed. Asynchronism and acceleration of polar activity are determined by the absence of the first cleavage. Chromosomal alterations (thickening, deformations and clumping) are partially reversible, but result in chromosomes breakage and recombinations. Stronger activity and toxicity of caprylic acid may be related to its longer carbonic chain.     

Action de dérivés du sélénium (séléniates et sélénites) comparée à celle d'acides divers (crotonique,acétique,citrique et phosphorique) sur les mitoses de segmentation

The action of seleniates and selenites on the segmentation mitoses is similar to that of SeO₂: polar dissociation with conserved dominance of the principal pole, stickiness and clumping of chromosomes. Crotonic and acetic acid dissociate more strongly the achromatic apparatus, which is resolved into divergent fibrillae, which are sinuous and irregularly fasciculated; the principal poles may be recognized, but their activity is more lessened: centrospheres are reduced to orangeophilic material. All these derivatives are acidic in the solutions. When the action of crotonic acid is interrupted, fibrillogenesis does not recover, but cellular centers start again to divide and the size of centrospheres enlarges by assemblage of dis-oriented cyanophilic material, arising from destroyed fibrillae. Reduction of fibrillogenesis by inactivation of the poles results in reactivation of fibrillogenesis in the diastema, expansion of the fibrillar ?clear zone? and its budding into numerous elements filling the animal hemisphere. Furrowing is consequently inhibited. Classification of mitotic dissociation is reviewed: equilibrated pluripolar mitoses (phenylurethane), pluripolar systems hierarchically organized (selenium dioxyde), pluripolar systems practically not hierarchically organized (crotonic and acetic acids).     

Partial disorganization of the anaphasic segregation of chromosomes in plant cells: combined actions of griseofulvin, producer of pluripolar anaphases and 2 ipecac alkaloids, producers of floating pole anaphases]

Anaphasis may be slightly checked by various treatments which however result in a normal chromosomic separation. Griseofulvin exerts a direct though partial influence on the mitotic apparatus, which entails "pluripolar anaphasis"; on the other hand Ipecac alkalo?ds act indirectly and produce "floating poles anaphases". Treatments combining griseofulvin with cepheline or tubulosine show that there is never any synergy between the two processes. These results support our hypothesis that floating poles anaphases are not a sign of slight C-mitotic action but only come from a lag between the appearance/disappearance of microtubules and that of chromosomes during anaphasis.     

Action of glutaraldehyde and formaldehyde on segmentation mitoses : Inhibition of spindle and astral fibres, centrospheres blocked

Glutaraldehyde and formaldehyde act as antimitotic substances at lower concentrations and as fixatives at higher ones. The arrested mitosis is of the quinoline type (d-mitosis); it is characterized by the disappearance of all the spindle and astral fibres, by the immobilization of chromosomes in the equatorial region and by the blocking of centrospheres. These blocked centrospheres are devoid of astral fibres, densified, and intensely stained; they do not diminish in volume at the end of mitosis. An explanation for this phenomenon is proposed: the binding of two or several microtubule subunits by one molecule of the active substance into the storage structures would prevent the discharge of these microtubule subunits and consequently the construction of microtubules. Colchicine-like substances bind only to one microtubule subunit and consequently storage structures cannot be formed. The relation between fixative and antimitotic properties is discussed. A gradient of sensibility to antimitotic action is observed: the mitoses are more easily arrested in the animal than in the vegetative hemisphere.     

Combinaison de deux actions antimitotiques différentes: dioxyde de sélénium et hydrate de chloral

Two antimitotic substances with different effects have been combined: the first, chloral hydrate M/10, destroys the achromatic fibers after a delay of about 1 h and a half to 2 hours; however, in this case chromosomes are not affected. The second, selenium dioxyde M/100, alters the chromosomes, without destroying the fibers, after a delay of forty five minutes to 1 h; however, it sometimes provokes the blockade of the centrosphere in the shape of a dense smooth bowl which is as voluminous as it is during the normal metaphase. When both substances are combined under conditions of maximum action, fibrillae are destroyed, but the centrosphere stays blocked. With chloral alone this blockade does not occur. As a consequence of the successive actions of chloral and selenium (each drug used respectively during half the time) the two primitive poles, which are still provided with inactive and fibrilless centrospheres, appear separated from a pluripolar spindle, the poles of which are active, but devoid of centrospheres. Therefore the action of selenium dioxide is qualitatively different from the action of chloral, and the various functions of the centriole (including gathering of the centrosphere material, fibrillogenesis and division of the center) could be separately damaged.     

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.     

Action de la quinoline sur les mitoses de segmentation des eufs d'urodèles: le blocage de la centrosphère

Quinoline in saturated solution (0,46 M) progressively destroys spindle and astral fibers, beginning in the juxtacentromeric region; it blocks the centrosphere material around the centriole. Chromosomes are immobilized in equatorial position far off the blocked centrospheres and may undergo telophasic transformation into karyomeres. Diastema may be inactivated before mitosis. Centrospheres are first deprived of some fibers, then granular and more or less dissociated, last completely smooth and segregated into cortex and medulla. Breaks and recombinations of chromosomes may appear after a long while, when a brief action is interrupted. With less concentrated solutions monopolar mitoses and monopolar telophases (rosettes) are observed (1/8 saturated solution), then shortened bipolar mitoses (1/16 saturated). Qualitative differences between quinoline and colchicine actions are evident.     

Open spindle nuclear division in the amoebal phase of the acellular slime moldEchinostelium minutum with chromosomal movement related to the pronounced rearrangement of spindle microtubules

Summary Myxamoebae ofEchinostelium minutum exhibit extranuclear (open spindle) mitosis with centrioles present at the poles. Spindle microtubules are formed in association with a juxtanuclear MTOC which surrounds the cell's complement of centrioles. During late prophase or prometaphase the nuclear envelope breaks down and subsequently a metaphase plate is formed. Two anaphasic movements occur sequentially: firstly, the distance of the chromosomes to the poles shortens; secondly the distance between the spindle poles increases. The arrangement of spindle microtubules during anaphase is consistent with the hypothesis that chromosomal separation is due to lateral interaction (zippering) of microtubules. During telophase, reconstitution of the nuclear envelope usually takes place in the interzonal region prior to reformation in the polar region. Cytokinesis, which begins in anaphase or early telophase involves the participation of vesicles, microfilaments and microtubules.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Microfilaments: dynamic arrays in higher plant cells

By using fluorescently labeled phalloidin we have examined, at the light microscope level, the three-dimensional distribution and reorganization of actin-like microfilaments (mfs) during plant cell cycle and differentiation. At interphase, mfs are organized into three distinct yet interconnected arrays: fine peripheral networks close to the plasma membrane; large axially oriented cables in the subcortical region; a nuclear "basket" of mfs extending into the transvacuolar strands. All these arrays, beginning with the peripheral network, disappear at the onset of mitosis and reappear, beginning with the nuclear basket, after cytokinesis. During mitotic and cytokinetic events, mfs are associated with the spindle and phragmoplast. Actin staining in the spindle is localized between the chromosomes and the spindle poles and changes in a functionally specific manner. The nuclear region appears to be the center for mf organization and/or initiation. During differentiation from rapid cell division to cell elongation, mf arrays switch from an axial to a transverse orientation, thus paralleling the microtubules. This change in orientation reflects a shift in the direction of cytoplasmic streaming. These observations show for the first time that actin-like mfs form intricate and dynamic arrays in plant cells which may be involved in many as yet undescribed cell functions.     

Transition from mitotic apparatus to cytokinetic apparatus in pollen mitosis of the slipper orchid

Summary Cytokinesis following asymmetrical pollen mitosis was studied in the slipper orchidCypripedium fasciculatum using techniques of immunofluorescence, confocal laser scanning, and transmission electron microscopy. Data from stereo reconstructions of double labelled preparations (microtubules/nuclei) show that the contribution of residual spindle fibers to development of the interzonal array is minor; rather, new populations of microtubules are nucleated in association with the two groups of anaphase chromosomes. As kinetochores reach the poles, trailing arms of the chromosomes and nonkinetochore microtubules are displaced outward in the equatorial zone and by early telophase the interzone is left virtually free of microtubules. The interzonal apparatus has its origin in a massive proliferation of microtubules from the polar regions and surfaces of contracting chromosomes. Each polar region appears as a hub from which microtubules radiate in a spoke-like configuration and numerous tufts of microtubules appear to emanate from margins of the chromosomes themselves. These newly organized arrays of microtubules extend to the equatorial region where they interact to form the interzonal apparatus. Increasing organization of microtubules in the interzone results in development of a typical phragmoplast configuration consisting of opposing cone-like bundles of microtubules bisected by an unstained equatorial line.     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.     

THE ULTRASTRUCTURE OF MITOSIS IN THE MARINE RED ALGA MEMBRANOPTERA PLATYPHYLLA1,2

Gametophyte germlings from unialgal cultures of Membranoptera platyphylla were examined with the electron microscope. The events of mitosis were observed in dividing cells near the thallus apex. In prophase the nucleus is spindle-shaped and surrounded by microtubules and a layer of endoplasmic reticulum. A unique organelle, the polar ring, is present at each pole; its junction is not clear. At metaphase the nuclear envelope is intact except for fenestrations at the poles. Spindle microtubules are attached to distinct kinetochores on the chromosomes and continuous pole-to-pole microtubules are present. The nucleolus has dispersed but, its granular components are still evident in the nucleoplasm. As the chromosomes separate, the nucleus elongates and finally constricts in the middle to produce 2 daughter nuclei.     

Ultrastructural disorders of the mitotic apparatus during cytochalasin B action

A correlation between the number of chromosome sets and the number of centrioles (8n--8 centrioles) was observed in polyploid metaphase cells, during cytochalasin B treatment on the cultured Chinese hamster cells. There is no correlation between the number of chromosome sets and the centriole number after stopping the action of the drug in many cells, but a great variation is observed in maintenance of chromosomes and centrioles (up 6 to 25 n and up 4 to 22 centrioles). In multipolar mitosis, either during the drug action or after its stopping, different numbers of chromosomes are directed towards the poles not depending on the number of centrioles in the poles. During the cytochalasin B treatment, either in bipolar or multipolar metaphases, there are destructions in the ultrastructure of the mitotic apparatus: there are no astral microtubules; in the poles there are diplosomes and duplex of centrioles with fibrillar material around both centrioles; kinetochores are of prometaphase type. After stopping the drug action the astral microtubules appear, but no other patterns of normalization in the mitotic apparatus occur. Desynchronization of three cycles (chromosomal, centriolar and centrosomal) is discussed as a factor of abnormal development of the mitotic apparatus and as a factor of stabilization of aneuploidy in the cell culture.     

Chromosome fiber dynamics and congression oscillations in metaphase PtK2 cells at 23 degrees C

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 microns or greater. DTAF-tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF-tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti-beta tubulin antibody. At 2-5 minutes after injection, concentrated DTAF-tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF-tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 microns) of concentrated DTAF-tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF-containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF-containing segments: 1) a poleward flux of tubulin subunits (Mitchison, 1989) or 2) capture of DTAF-containing nonkinetochore microtubules.     

Stable versus unstable orientations of sex chromosomes in two grasshopper species

The structural basis of orientation stability was investigated. The stable unipolar orientation of the Melanoplus sanguinipes X-chromosome univalent is unique in that it is stable without tension created by forces towards opposite poles; tension is thought to be the principle component in stabilizing kinetochore orientations to a pole. Stable orientation of the X chromosome in Melanoplus sanguinipes was compared with unstable X orientation in Melanoplus differentialis. Ten cells (five of each species) were studied, firstly in living cultures where chromosome behavior was followed, then by serial-section electron microscopy where the structural basis for chromosome behavior was examined. Microtubules other than kinetochore microtubules were observed impinging on the X chromosomes. One end of these microtubules was buried in chromatin, while the other ran towards a pole. The X chromosomes of M. sanguinipes had more of these microtubules than did M. differentialis X chromosomes. It is suggested that M. sanguinipes X chromosomes are less condensed than M. differentialis X chromosomes and so allow more microtubules to penetrate the chromosome. The extra microtubules impinging on the M. sanguinipes X chromosome probably prevent reorientation by inhibiting the turning of the chromosome towards the opposite pole, i.e., more force is needed to turn a kinetochore towards the opposite pole than can be generated and attempts at reorientation fail. This may be analogous to the effect that tension has on the orientation stability of bivalents.     

Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol

Fluorescently labeled tubulin was quickly incorporated into the mitotic apparatus when injected into a live sand dollar egg. After a rectangular area (1.6 X 16 microns) of the mitotic spindle was photobleached at metaphase or anaphase by the irradiation of a laser microbeam, redistribution of fluorescence was almost complete within 30 sec. The photobleached area did not change in shape during the redistribution. During the period of redistribution, the bleached area moved slightly toward the near pole at metaphase and anaphase (means: 1.6 and 1.8 micron/min, respectively). These results indicate that redistribution was not due to the exchange of tubulin subunits only at the ends of microtubules but to their rapid exchange at sites along the microtubules in the bleached region. Furthermore, treadmilling of tubulin molecules along with the spindle microtubules possibly occurred at the rate of 1.6 micron/min at metaphase. Birefringence of the mitotic apparatus increased with a large increase in both the number and length of astral rays shortly after taxol was injected. However, the microtubules did not all seem to elongate at the same rate but appeared to become equalized in length. Chromosome movement stopped within 60 sec after the injection. Centrospheres became large and the labeled tubulin already incorporated into the centrospheres was excluded from the enlarged centrospheres. Shortly after the labeled tubulin was injected following the injection of taxol, it accumulated in the peripheral region of the centrospheres, suggesting that microtubules first assembled at this region. Fluorescently labeled tubulin in the mitotic apparatus in the egg after injection of taxol was redistributed much more slowly after photobleaching than in uninjected eggs.     

Aster formation in vitro is nucleated by granules isolated from the mitotic apparatus

Mitotic apparatuses (MAs) isolated from sea urchin eggs contained clusters of granular material in their centrospheres. After cold treatment and mild agitation, the MA fraction formed asters when combined with tubulin. Many microtubules grew from isolated centrospheres most of which were covered with astral residues. Homogenization of the isolated MA fraction dispersed the centrospheres which broke into fragments or into aggregates of small granules that formed small asters when tubulin was added. Electron microscopy showed that more than ten microtubules were nucleated from a granular aggregate composed of several approximately 90-nm granules. The aster-forming activity was lost with time when the MAs were kept at 0 degree C. Only glycerol stabilized this activity. The aster-forming activity also was heat labile and trypsin sensitive, but it was resistant to RNase treatment. When the dispersed MAs were extracted with a buffer solution of high ionic strength, aster-forming activity was recovered only in the extract; that is, when the extract had been dialyzed against a solution of low ionic strength, the fine granules self assembled and retained their aster-forming ability.     

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.