Mitosis of the free-living flagellate Bodo saltans strain Ps+ (Kinetoplastidea, Bodonida)

Mitosis of the free-living flagellate Bodo saltans of the Ps+ strain characterized by the presence of prokaryotic cytobionts in the perinuclear space was studied. Division of B. saltans Ps+ nuclei occurs by the closed intranuclear type of mitosis without condensation of chromosomes. At the initial stages of nuclear division, consecutive anlage of two spatially separated microtubular spindles begins. The spindle containing about 20 microtubules appears first, then, at an angle of 30–40° to it, the second spindle containing half as many microtubules is formed. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one—with 2 pairs. The kinetochores of B. saltans Ps+ have a pronounced laminar structure. Both spindles rest with their ends directly on the internal membrane of the nuclear envelope and form 4 well-pronounced poles. The equatorial phase of mitosis in B. saltans Ps+ is not revealed. The divergence of sister kinetochores towards the poles occurs independently in each spindle. At the elongation phase of mitosis, the poles of both spindles are united in pairs to form a single bipolar structure composed of two loose bundles of microtubules. At this stage of nuclear division, the kinetochores reach the poles of the subspindles and cease to be visible. At subsequent nuclear division stages the nucleus acquires a dumbbell shape. During the reorganization phase the sister nuclei are separated. In the perinuclear space of the interphase nuclei of B. saltans Ps+, 1–2 prokaryotic cytobionts are present. In the course of mitosis, these organisms divide intensively, such that their number can reach 20 and more per nucleus. During separation of sister nuclei, the “excessive” cytobionts are released into the cytoplasmic vacuoles formed by external membranes of the nuclear envelope.     

MITOSIS IN THE FUNGUS THRAUSTOTHECA CLAVATA

The ultrastructure of mitosis is described in Thraustotheca clavata, an oömycete fungus. An intranuclear spindle develops between differentiated regions of the nuclear envelope which move apart, each associated with 180° oriented centriole pairs. The spindle contains low numbers of continuous and interdigitating microtubules in addition to chromosomal microtubules. Each kinetochore is attached to only one microtubule. Serial section analysis shows that at meiosis there are probably 12 chromosomes in the diploid nucleus, yet at mitosis the methods utilized in the present study suggest that there may be less than 12 kinetochores connected to each pole. At mitosis many of the kinetochores within a given spindle are not arranged in opposite pairs. The behavior of the spindle microtubules during mitosis is comparable to that of higher organisms but the rarity of short intertubular distances appears to preclude significant force generation by means of intertubular bridge mechanisms. Evidence is presented for a nuclear envelope-microtubule interaction which is capable of generating shear forces during both mitosis and interphase nuclear movements.     

Mitosis in the free-living flagellate Bodo saltans strain PS+ (Kinetoplastidea, Bodonida)

The mitosis in the free-living flagellate Bodo saltans Ps+ with prokaryotic cytobionts in perinuclear space has been studied. The nuclear division in B. saltans Ps+ occurs by closed mitosis type without condensation of chromosomes. Two spatially separated mitotic spindles begin to form consistently at the initial stages of nuclear division. The spindle including about 20 microtubules appears first and later the second spindle with half the number of microtubules comes at the angle of 30-40 degrees. Both spindles rest their ends against the inner nuclear membrane and form 4 distinct poles. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one are associated with 2 pairs of kinetochores. The divergence of the kinetochores towards the poles occurs independently in each spindle. The equatorial phase is not revealed in B. saltans Ps+. The poles of both spindles unite in pairs at the elongation phase of mitosis and form the integrated bipolar structure. At this stage of the nuclear division, the kinetochores reach the poles of subspindles and become indistinguishable. Then the nucleus takes the shape of a dumbbell. The inner nuclear membranes of just formed nuclei have layers of condensed chromatin characteristic of the interphase nuclei of kinetoplastidea. The daughter nuclei separate at the phase of reorganization. There are 1-2 prokaryotic endocytobionts in the perinuclear space of the interphase nuclei in B. saltans Ps+. The symbionts multiply during mitosis and their number reaches more than 20 specimens par nucleus.     

An electron microscope study of nuclear and cell division in a dinoflagellate

Summary Light microscopical observations on the cell division of the small dinoflagellate Woloszynskia micra are correlated for the first time with an electron microscopical study. In prophase, whilst the nucleus enlarges and becomes pearshaped, the chromosomes divide to give pairs of chromatids. This process starts at one end and works to the other giving Y- and V-shaped chromosomes as it occurs. Cytoplasmic invaginations pass through the nucleus and by the end of prophase these are seen to contain a number of microtubules of about 180 Å diameter. There is no connection between the microtubules in the nuclear in vagination and either the flagellar bases or the chromosomes. At anaphase the nucleus expands laterally and the sister chromatids move towards opposite ends. The cell hypocone is now partially divided and the two longitudinal flagella well separate. The nucleus completes its division into two daughter nuclei and for a time portions of the cytoplasmic invaginations remain visible. Cell cleavage is completed by the division of the epicone. The nuclear membrane remains intact throughout division and the nucleolus does not break down.The mitotic division in this organism, which is unusual in comparison with the mitosis of higher organisms, is discussed in the light of other types of mitosis which have been reported and of earlier light microscopical observations on dinoflagellates.     

THE FINE STRUCTURE OF MITOSIS AND CELL DIVISION IN THE CHRYSOPHYCEAN ALGA OCHROMONAS DANICA1

At the ultrastructural level, cell division in Ochromonas danica exhibits several unusual features. During interphase, the basal bodies of the 2 flagella replicate and the chloroplast divides by constriction between its 2 lobes. The rhizoplast, which is a fibrous striated root attached to the basal body of the long flagellum, extends under the Golgi body to the surface of the nucleus in interphase cells. During proprophase, the Golgi body replicates, apparently by division, and a daughter rhizoplast, appears. During prophase, the 2 pairs of flagellar basal bodies, each with their accompanying rhizoplast and Golgi body, begin to separate. Three or 4 flagella are already present at this stage. At the same time, there is a proliferation of microtubules outside the nuclear envelope. Gaps then appear in the nuclear envelope, admitting the microtubules into the nucleus, where they form a spindle. A unique feature of mitosis in O. danica is that the 2 rhizoplasts form the poles of the spindle, spindle microtubules inserting directly onto the rhizoplasts. Some of the spindle microtubules extend from pole to pole; others appear to attach to the chromosomes. Kinetochores, however, are not present. The nuclear envelope breaks down, except, in the regions adjacent, to the chloroplasts; chloroplast ER remains intact throughout mitosis. At late anaphase the chromosomes come to lie against part of the chloroplast ER. This segment of the chloroplast ER appears to be incorporated as part of the reforming nuclear envelope, thus reestablishing the characteristic nuclear envelope—chloroplast ER association of the interphase cell.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

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.     

Mitosis in the cellular slime mold Polysphondylium violaceum

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re- establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.     

Ultrastructure of the micronucleus of the infusorian Didinium nasutum during meiosis]

Six stages can be distinguished in the micronuclear first maturation division prophase of D. nasutum. Nucleolus-like structures of fibrillar nature, connected with micronuclear chromosomes seem to develop at the late leptotene. At zygotene-pachytene, the chromosomes condense, forming irregular loops. This coincides with formation of classically structured synaptinemal complexes in the micronuclei. At diplotene-diakinesis, chromosomal bivalents are uniformly scattered throughout the micronucleus. They aggregate into a net equatorial plate in the first division metaphase; chromosomes show prominent kinetochores with attached chromosomal microtubule bundles. The second maturation division starts immediately after the completion of the first division and is morphologically similar to agamic mitosis of the micronuclei of D. nasutum. During the 2th maturation division prophase, the compact chromosomes form a dense group and show no spreading inside the nucleus. They are interspaced by an amorphous material being possibly involved in the formation of spindle microtubules. The telophase spindle of the 2nd division likely as that of the Ist division divides into three parts, the two daughter nuclei and the separation spindle containing a material of depolymerized microtubules. Only one of the 2nd division derivatives enters the third maturation division. A short telophasic third division spindle is perpendicular to the surface of the contact between the conjugants and produces two pronuclei. The envelopes of the daughter micronuclei are formed from parts of the original nuclear envelope surrounding the entire spindle.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.     

Ultrastructure of mitosis in the aphid-pathogenic fungusErynia neoaphidis

Summary An account of mitosis in the aphid-pathogenic, entomophthoraceous fungusErynia neoaphidis is presented. The mitotic apparatus is characterized by a closed, intranuclear, polarized spindle. Chromosomes are permanently attached by kinetochore microtubules (kcMTs) to the poles during mitosis. The spindle develops as the spindle pole bodies migrate and separate. At metaphase the eccentric spindle contains only kcMTs and is located in a relatively chromatinfree zone. Paired sister kinetochores are arranged in a broad metaphase plate. During anaphase kcMTs shorten, astral and nonchromosomal microtubules develop and elongate and the interpolar distance increases.     

Holokinetic composite chromosomes with “diffuse” kinetochores in the micronuclear mitosis of a heterotrichous ciliate

During micronuclear mitosis of the heterotrichous ciliate Nyctotherus ovalis Leidy rod-shaped composite chromosomes are formed by lateral association of telokinetic chromosomes. The formation of these composite chromosomes seems to be a highly ordered process since only nuclei with either 18 or 24 such chromosomes can be observed, and nuclei with the same chromosome number show a similar length distribution of their chromosomes. Further, these data indicate that we examined two otherwise indistinguishable races. During metaphase the composite chromosomes become arranged in the spindle equator in a holokinetic fashion, their entire poleward surfaces being covered by kinetochore material. These diffuse kinetochores have a trilaminar appearance comparable to those of monokinetic chromosomes. Their electron density after employing Bernhard's procedure revealed the same ribonucleoprotein distribution as reported for the localized kinetochores formed during the extranuclear mitosis in other cells. During early anaphase the outer kinetochore layer remains continuous while the individual chromosomes in the composite group show a tendency to separate leaving chromatin-free spaces of about 40 nm diameter. Kinetochore microtubules which are still anchored in the outer kinetochore layer seem to elongate and to extend into the interpolar spindle region predominantly through these holes in the chromatin. These observations suggest a like polarity of kinetochore and interpolar microtubules in the polar spindle region while microtubules in the interpolar space seem to interdigitate in an antiparallel fashion. The activity of the kinetochore to act as a microtubule-organizing center (MTOC) seems to be modulated by the chromatin underlying the outer kinetochore layer which may prevent further outgrowth of kinetochore microtubules.     

The ultrastructure of mitosis inChroomonas salina (Cryptophyceae)

Summary A detailed account of the ultrastructure of mitosis in a member of theCryptophyceae is given for the first time. The initial indication of mitosis is the duplication of the flagellar bases. The nucleus migrates towards the anterior of the cell and its envelope and nucleolus break down. The chromatin which at interphase is in the form of scattered clumps, condenses into a solid mass through which run narrow tunnels. Each tunnel allows the passage of one to four microtubules. At metaphase the dense plate of chromatin is situated on the equator and the spindle has a rectangular shape. Individual chromosomes cannot be recognized and no morphologically differentiated kinetochores have been observed. The flagella remain functional, their bases stay at the anterior side of the nucleus and do not move to the poles. At anaphase two plates of chromatin separate and these move apart until they come to lie against the ER sheath surrounding the chloroplasts. The new nuclear envelope starts to form on the opposite side of the daughter nucleus. Cytokinesis may commence early in mitosis and consists of a constriction of the parent cell, starting from the posterior end, followed by separation of the two daughters. The present work supports earlier views that only one chromosome is evident during the nuclear division of these organisms. The mitosis is completely different from that of theDinophyceae with which theCryptophyceae were formerly linked.     

A UNIQUE MITOTIC VARIATION IN THE MARINE DINOFLAGELLATE OXYRRHIS MARINA (PYRROPHYTA)1

Mitosis is described in the flagellate Oxyrrhis marina Dujardin and is compared in related genera. Dense plaques develop in the nuclear envelope at prophase and give rise to an intranuclear spindle. Some of the microtubules associate with the chromosomes while others extend across the nucleus. The basal bodies migrate toward the poles early in division and retain a position lateral to the nuclear poles throughout mitosis. Microtubules are not present between the nucleus and the basal bodies. The nucleolus is persistent and elongates throughout anaphase and telophase. Chromosomal separation is accomplished by sliding of non-chromosomal microtubules and by elongation of the nuclear envelope rather than by shortening of the spindle microtubules. The nuclear envelope begins to constrict in the center early in anaphase. Continued constriction of the envelope and elongation of the nucleus leads to the formation of a dumbbell-shaped nucleus by late telophase. Mitosis culminates by the constriction of the nucleus into two daughter nuclei. The taxonomic position of Oxyrrhis marina is discussed in light of these findings.     

ULTRASTRUCTURE OF CELL DIVISION AND REPRODUCTIVE DIFFERENTIATION OF MALE PLANTS IN THE FLORIDEOPHYCEAE (RHODOPHYTA): CELL DIVISION IN POLYSIPHONIA1

Cell division in the marine red algae Polysiphonia harveyi Bailey and P. denudata (Dillwyn) Kutzing was studied with the electron microscope. Cells comprising the compact spermatangial branches of male plants were used exclusively because of their small size, large numbers and the ease with which the division planes can be predetermined. Some features characterizing mitosis in Polysiphonia confirm earlier electron microscope observations in Membranoptera, the only other florideophycean algae in which mitosis has been studied in detail. Common to both genera are a closed, fenestrated spindle, perinuclear endoplasmic reticulum, a typical metaphase plate arrangement of chromosomes, conspicuous, layered kinetochores, chromosomal and non-chromosomal microtubules, and nucleus associated organelles (NAOs) known as polar rings (PRs) located singly in large ribosome-free zones of exclusion at division poles in late prophase. However, other features, unreported in Membranoptera, were observed consistently in Polysiphonia. These include the presence of PR pairs in interphase-early prophase cells, the attachment of PRs to the nuclear envelope during all mitotic stages, the migration of a single PR to establish the division axis, a prominent, nuclear envelope protrusion (NEP) at both division poles at late prophase, the prometaphase splitting of PRs into proximal and distal portions, and the reformation of post-mitotic nuclei by the separation of an elongated interzonal nuclear midpiece at telophase. During cytokinesis, cleavage furrows impinge upon a central vacuolar region located between the two nuclei and eventually pit connections are formed in a manner basically similar to that reported for other red algae. Diagrammatic sequences of proposed PR behavior during mitosis are presented which can account for events known to occur during cell division in Polysiphonia. Mitosis is compared with that reported in several other lower plants and it is suggested that features of cell division are useful criteria to aid in the assessment of phylogenetic relationships of red algae.     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs microtubules     

Electron microscopic studies on the foraminiferAllogromia laticollaris arnold mitosis in agamonts

Summary During mitosis in multinucleated agamonts ofAllogromia laticollaris the nucleolar substance cannot be demonstrated in the nuclei, but only in the cytoplasm as large opaque bodies, some of which are surrounded by two membranes. Only a few smaller bodies are still present within vacuoles of young agamonts formed subsequent to the dividing state. Since the nuclear envelope persists during karyokinesis, the division spindle is localized intranuclearly. The component continuous microtubules, which prefer the nuclear periphery, are mostly visible as bundles, whereas the microtubules running to the chromosomes appear single and less numerous. Centrosomes can be noticed not only at elongated but also at ovoid or irregularly shaped nuclei. These centrosomes represent round or elliptic bodies surrounded by a membrane and consist of granular and fibrillar material. They occur within the perinuclear space and are restricted to the state of karyokinesis.     

Primitive mitotic mechanisms.

Unorthodox mitotic mechanisms are reviewed and their contribution to the understanding of evolution of the orthodox mitotic apparatus is considered. Dinoflagellates and hypermastigote flagellates are of particular significance because the microtubular mitotic apparatus is entirely extranuclear with the nuclear membrane persisting through mitosis. Chromosomes are attached to the nuclear membrane. In hypermastigole flagellates early kinetochore separation is on the nuclear membrane without any contribution from microtubules. In dinoflagellates the chromosomes are also attached to the nuclear membrane, but at least in some species cytoplasmic microtubules connect to the attachment site. In Syndinium the attachment site resembles a typical kinetochore, but is inserted in the nuclear membrane. A similar kinetochore is found in certain Radiolaria, but with an intranuclear spindle apparatus the association with the nuclear membrane is no longer necessary and has been lost. Mitosis in the yeast Saccharomyces is essentially orthodox, though chromosomes do not condense. No kinetochores are seen, but a single microtubule makes direct contact with the 20 nm chromatin fiber of each chromosome and shortens during anaphase. About 5-10 microtubules are continuous between the spindle pole bodies and form the elongating central spindle.     

ULTRASTRUCTURE AND TIME COURSE OF MITOSIS IN THE FUNGUS FUSARIUM OXYSPORUM

Mitosis in Fusarium oxysporum Schlect. was studied by light and electron microscopy. The average times required for the stages of mitosis, as determined from measurements made on living nuclei, were as follows: prophase, 70 sec; metaphase, 120 sec; anaphase, 13 sec; and telophase, 125 sec, for a total of 5.5 min. New postfixation procedures were developed specifically to preserve the fine-structure of the mitotic apparatus. Electron microscopy of mitotic nuclei revealed a fibrillo-granular, extranuclear Spindle Pole Body (SPB) at each pole of the intranuclear, microtubular spindles. Metaphase chromosomes were attached to spindle microtubules via kinetochores, which were found near the spindle poles at telophase. The still-intact, original nuclear envelope constricted around the incipient daughter nuclei during telophase.     

Progress in studies of ZW10, a proper chromosome segregation protein

The conserved protein ZW10 is found in various organisms. It is localized on the kinetochores or spindle microtubules during cell division. ZW10 regulates not only the segregation of homologous chromosomes, each consisting of attached sister chromatids (during the first meiotic division), but also the separation of individual chromatids (during mitosis and the second meiotic division). ZW10 is required for proper chromosome segregation during both mitosis and meiosis. The effects of zwl0 mutations are similar for both equational and reductional divisions, giving rise to anaphases with lagging chromosomes and/or unequal numbers of chromosomes at the two poles. The localization of ZW10 is similar during mitosis, meiosis I, and meiosis II. In interphase the distribution of ZW10 changes; it is localized in the endoplasmic reticulum, Golgi apparatus, and in the cytosol and is involved in membrane trafficking between the endoplasmic reticulum and Golgi apparatus. ZW10 forms a subcomplex with RINT-1 and p31 which are involved in a larger complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE that is implicated in membrane trafficking. The text was submitted by the authors in English.