Mitotic Centers Research Articles

In situ analysis of cultured Potorous cells. I. DNA synthesis and mitotic dynamics in bi- and multinucleate cells.

The rat-kangaroo (Potorous tridactylis) cell line Pt-Kl was analyzed in situ after continuous labeling with H3-thymidine for 4–12 hours. Both DNA synthesis and early stages of mitosis were found to be incompletely synchronous in bi- and multinucleate cells. Chromosomal complements, however, catched up with each other so that the trigger of anaphase affected all chromosomes simultaneously. In these cells mitosis was usually multipolar and occasionally bipolar. Both S and mitosis were of longer duration than in mononucleate cells. Apparently, each nucleus enters mitosis with its own mitotic centers and interactions and coordinations among chromosomes and centers lead to the formation of spindle connections between related and unrelated poles, thus giving rise to a multipolar spindle which is a functional unit. Chromosomes from different nuclear origins do not mix with each other on the common bi- or multipolar equatorial plate. In binucleate cells undergoing quadripolar mitosis, each chromosomal complement was usually oriented on two adjacent arms of the X-shaped equatorial plate. The four daughter nuclei thus represented two parental and two recombinant types. The labeling patterns of recombinant nuclei indicated that chromosomes maintain their positions during the transition from anaphase to interphase. The implications of the present findings were discussed in relation to other related phenomena such as somatic segregation, chromosome behavior in hybrid cells, origin of haploid and triploid cells in diploid cell populations, depolyploidization, spatial relationships among chromosomes in bi- and multipolar spindles, and the orderly arrangement of interphase chromatin.

Microinjected carboxylated beads move predominantly poleward in sea urchin eggs.

Observations on living mitotic cells have suggested that material in the spindle moves poleward during mitosis. In order to investigate this movement, sea urchin eggs have been microinjected with 0.25-micron diameter carboxylated fluorescent beads. When fluorescent beads were injected into unfertilized Lytechinus variegatus eggs, no motility was detected. When injected into mitotic cells, beads moved to the spindle poles. Individual beads moved rapidly, in a saltatory fashion, and followed generally linear paths. Beads appeared to move along astral fibers, were generally excluded from the spindle proper, and accumulated at the spindle poles. Some dispersion of the beads away from the pole was observed as cells completed mitosis, but the majority of beads retained a polar location. After depolymerization of spindle microtubules with nocodazole, some dispersion of beads into the cytoplasm was also observed. Beads moved along taxol-induced astral microtubules and accumulated at astral centers. These observations reveal that negatively charged beads accumulate rapidly at mitotic centers, moving toward the minus end of the microtubules. Neither the bidirectional motility of similar beads in interphase cells nor the plus-end-directed bead motility seen in axons was observed in these mitotic cells.

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs: (astral rays/fertilization/mitosis/microinjected polystyrene beads/sand dollar eggs).

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis. Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2-0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.

Indirect immunofluorescent staining of the microtubules in interphase and mitotic amoebae of the myxomycetePhysarum polycephalum

The microtubules ofPhysarum amoebae have been decorated with rat antibodies against yeast tubulin. The indirect fluorescent staining observed in interphase amoebae and in flagellated amoebae is consistent with the three-dimensional reconstructions previously deduced from electron microscopic studies. Mitotic amoebae exhibit a pattern of fluorescence which is similar to that exhibited by mammalian cells and is consistent with the previous electron microscopic studies, except that we also observe pole-pole microtubule fibers during metaphase and anaphase and the presence of a typical midbody during cytokinesis. The various types of tripolar mitosis which are observed suggest that there is a regulatory mechanism allowing the formation of pseudo-bipolar mitotic apparatuses in amoebae possessing more than two mitotic centers during mitosis. The mitotic center, located in the middle of the centrosphere, is not fluorescent after staining of the monoasters induced with taxol suggesting the absence of tubulin in the mitotic center.

Microtubule polarity and spindle function

The idea that microtubule (MT) polarity is important for spindle function was first proposed more than a decade ago. However, it was not until 1974 that this idea could be put to a direct test. The identification of methods for MT polymerization in vitro made it possible to use flagella and cilia as seeds for purified brain tubulin; it was demonstrated that under given conditions one end of the MT grows at a faster rate than the other. The two different ends were designated 'plus' and 'minus' end. Subsequently efforts were made to determine the polarity of spindle MTs with the help of these rate measurements. Mitotic centers as well as chromosomes were isolated and the rate of MT growth from these initiation sites was compared to the rates of MT growth from the two ends of flagella or cilia. The results from these experiments revealed that MTs were growing at a fast rate from both structures, suggesting that the plus ends of aster and kinetochore MTs are distal to these sites.

Caffeine-induced monaster cycling in fertilized eggs of the sea urchin Strongylocentrotus purpuratus

Caffeine has several highly visible effects on mitosis in eggs of the sea urchin Strongylocentrotus purpuratus. It increases the cell cycle length in a dose-dependent manner and at a concentration between 5 and 10 m M it prevents the normal separation and function of the mitotic centers, resulting in periodic monaster formation rather than division. If fertilized eggs are placed in seawater containing 10 m M caffeine at prometaphase, the spindle rapidly shrinks, moving the mitotic centers to the middle of the metaphase plate in less than 10 min. Within 30 min the eggs recover their ability to polymerize microtubules to form a large monaster, and from this time on they continue to cycle monasters in the presence of caffeine, with a period of about 150 min, where the normal second division time is 55 min. Other cell cycle functions, such as chromosome condensation, nuclear membrane breakdown and reformation, attempted furrowing and surface membrane addition continue for at least several cycles, but at the slower rate. Although the evidence strongly suggests that the rapid breakdown of the prometaphase spindle results from a calcium release induced by caffeine, these experiments do not rule out other physiological effects of caffeine on the longer-term changes in cycle length, centriole function, and ultimately on cell survival.

Spatial relationships between centrioles and the centrosphere in monoasters induced by taxol inPhysarum polycephalum amoebae

Monoasters induced by taxol in the amoebae of the MyxomycetePhysarum polycephalum show an unusual tridimensional location of the centrioles. Tridimensional reconstructions of individual monoasters with either two or four centrioles show that the position of centrioles is not random. The characteristics of these monoasters suggest that the centrioles are not linked to the mitotic center in the monoaster since mitotic centers completely devoid of centrioles in adjacent or central location are observed. However, the preferential centrifugal orientation of the centrioles in the centrosphere induced by taxol suggests that centrioles are initially located in the mitotic center in agreement with the attachment of the centrioles to the mitotic center during interphase.

A quantitative analysis of mitotic gradients during early development in Calliphora erythrocephala Meig. (Diptera).

Mitotic waves during superficial cleavage and early gastrulation were analyzed quantitatively in Calliphora. Three consecutive patterns are present: (1) a monotonic anterioposterior mitotic gradient during early superficial cleavage; (2) a double mitotic gradient from the anterior and posterior poles during superficial cleavage, especially toward the end of the period; and (3) more complicated patterns with intermediate mitotic centers during the last superficial cleavage division and during early gastrulation. Mitotic gradients are absent in many eggs during early superficial cleavage, but they then become ubiquitous. The gradients are longitudinal; no transverse component was detected before gastrulation. Anterior and posterior gradient patterns are not mirror images of each other; mitotic activity always starts earlier anteriorly. The gradients are accompanied by a pronounced increase in interphase length. The mitotic gradients are compared with the morphogenetic gradients predicted in a current model for pattern specification in insect eggs.

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells.

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.

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.

Centriole behavior in chloramphenicol-treated eggs of the sand dollar, Dendraster excentricus.

This electron microscope study was undertaken to test the prediction made from an indirect assay method for mitotic centers (centrioles), that chloramphenicol inhibits centriole replication during first cleavage division in the eggs of the sand dollar,Dendraster excentricus. Extensive serial sectioning through both untreated and chloramphenicol-treated eggs, coupled with thorough examination of these sections, has demonstrated that 57% of the untreated eggs and 14% of the chloramphenicol-treated eggs contained paired centrioles at a time when centriole pairs normally exist. This study thus gives direct evidence that chloramphenicol inhibits centriole replication.

Bipolar and tetrapolar cleavage time in sea urchin eggs

The cleavage time of sea urchin eggs which were induced to divide into four cells by polyspermy and mercaptoethanol treatment was compared with the division into two cells. Tetrapolar division in eggs treated with mercaptoethanol occurred earlier than bipolar division. This result is consistent with the hypothesis of a diffusible substance from the mitotic centers which induces the cortex to form the cleavage furrow.

An indirect method to assay for mitotic centers in sand dollar (Dendraster excentricus) eggs.

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C(14)-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.

CLEAVAGE OF THE SEA URCHIN EGG RECOVERED FROM THE CLEAVAGE-BLOCKING EFFECT OF DEMECOLCINE***

EmbryologiaVolume 7, Issue 1 p. 68-80 Free Access CLEAVAGE OF THE SEA URCHIN EGG RECOVERED FROM THE CLEAVAGE-BLOCKING EFFECT OF DEMECOLCINE*** NAOMASA KOBAYASHI, NAOMASA KOBAYASHI Zoological Institute, College of Science, University of Kyoto, KyotoSearch for more papers by this author NAOMASA KOBAYASHI, NAOMASA KOBAYASHI Zoological Institute, College of Science, University of Kyoto, KyotoSearch for more papers by this author First published: January 1962 https://doi.org/10.1111/j.1440-169X.1962.tb00160.xCitations: 7 * Contributions from the Seto Marine Biological Laboratory, No. 368. ** This work was supported by a grant in Aid for Fundamental Scientific Research of the Ministry of Education. Nos., 4066-4068. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Literature Beams, H. W. and T. C. Evans, 1940. Some effects of colchicine upon the first cleavage in Arbacia punctulata. Biol. Bull., 79, 188– 198. Gray, J., 1931. A text-book of experimental cytology. Cambridge University Press, London . Harvey, E. B., 1927. The effect of lack of oxygen on sea urchin eggs. Biol. Bull., 52, 147– 160. Kobayashi, N., 1961. Effect of demecolcine upon cleavage of sea urchin eggs and their reversibility. Mem. Coll. Sci. Univ. Kyoto, Ser. B, 28, 1– 9. Mazia, D., 1958. SH compounds in mitosis. I. The action of mercaptoethanol on the egg of the sand dollar Dendraster excentricus. Exp. Cell Res., 14, 486– 494. Mazia, D., and A. M. Zimmerman, 1958. SH compounds in mitosis. II. The effect of mercaptoethanol on the structure of the mitotic apparatus in sea urchin eggs. Exp. Cell Res., 15, 138– 153. Mazia, D., and P. J. Harris and T. Bibring, 1960. The multiplicity of the mitotic centers and the time-course of their duplication and separation. J. Bio-phys. Biochem. Cytol., 7, 1– 20. Wilson, E. B., 1902. Experimental studies in cytology. II. Some phenomena of fertilization and cell-division in etherized eggs. III. The effect on cleavage of artificial obliteration of the first cleavage-furrow. Arch. Entw. Mech., 13, 353– 395. Citing Literature Volume7, Issue1January 1962Pages 68-80 ReferencesRelatedInformation

The Multiplicity of the Mitotic Centers and the Time-Course of Their Duplication and Separation

In this study, the reproduction of the mitotic centers in the eggs of a sea urchin, Strongylocentrotus purpuratus and a sand dollar Dendraster excentricus has been studied by means of experimental designs that do not depend on the actual visualization of centrioles. The centers are defined in operational terms as potential poles. Blockage of mitosis by mercaptoethanol, it was found, inhibits the duplication of the centers, but does not inhibit the splitting and separation of centers that have already duplicated and thus potential poles could be realized as actual poles in multipolar divisions. At all times, the center is at least a duplex structure; that is, it contains two potential poles. The actual duplication process is the earliest event in a given mitotic cycle, taking place at very early interphase or in late telophase of the previous division. The splitting of the centers following duplication is a distinct process, dissociable from the duplication as such. Duplication and splitting normally occur at about the same time in the mitotic cycle, with a precession of the former. That is, as the two members of a pair of "old" centers split, each one gives rise to a new one, which remains associated with it until the next phase of splitting and duplication occurs. The results are consistent with what is termed a "generative" model of the self-reproduction of an intracellular body. According to this, the body does not immediately produce a full-fledged copy of itself, with simultaneous fission, but the primary duplication event involves only a part of the parent structure. This gives rise to a "germ" or "seed" which then grows to be equivalent to the parent body, and finally splits from it.

The organization of the early embryo of bufo cognatus as revealed especially by the mitotic index

The present study has shown the following facts concerning the early embryology of Bufo cognatus: 1. In their essentials, the descriptive phases of the early embryonic development follow the well known amphibian pattern. 2. The mitotic index decreases as development progresses. There is a very sudden and marked decline soon after the onset of gastrulation. 3. Gastrulation is the most important stage in the organization of the embryo. During this time, important centers of proliferation are set up which produce cells concerned in the organization. 4. Epiboly is involved only to a limited extent in gastrulation. The endoderm arises mostly from heavily yolk-laden cells. 5. The notochord comes neither from endoderm as such nor mesoderm as such but is produced with these from a mass of rapidly dividing cells of the yolk region designated here as endo-chordo-mesoderm. 6. Cell movements from centers of mitotic activity play a major part in the organization of the embryo. 7. Specifically, ten centers of mitotic activity have been recognized each of which produces cells which move to a fairly well defined region. These centers have been shown to produce cells which later form (a) the neural tube, (b) most of the endoderm, (c) most if not all of the mesothelium, and (d) the notochord. In addition certain of them contribute to the market apparent lengthening of the body which begins directly after the blastopore closes. 8. The two most striking things shown are (a) that there is often a marked asymmetry in the position of the mitotic centers (as was also found in a teleost fish by Self, 1937) and (b) that internal movements of cells as well as the external movements shown by others for various Amphibia are involved in a very important way in the early organization of an amphibian embryo.