TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

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.     

Ultrastructural Features of Mitosis in Anisonema sp. (Euglenida)1

The fine structure of stages in mitosis in a colorless euglenoid, Anisonema sp., reveals that chromosomes remain condensed throughout the life cycle and are attached to the nuclear envelope at interphase. The onset of mitosis is marked by the anterior migration of the nucleus towards the base of the reservoir and by elongation of the nucleolus. The nuclear envelope persists throughout mitosis. Microtubules are generated in the peripheral nucleoplasm adjacent to the envelope and attach to the chromosomes while they are still associated with the envelope. The region of microtubular contact develops into a distinct layered kinetochore as the developing spindle with attached chromosomes separates from the nuclear envelope and moves into the nucleoplasm. The mature spindle consists of a number of subspindles each containing about 8–10 microtubules and a few associated chromosomes. Both chromosomal and non-chromosomal microtubules are present in each subspindle and extend towards the envelope terminating at or near the nuclear pores. Chromosomal segregation is concomitant with nuclear elongation. By late division, an interzonal spindle develops in the dumbbell-shaped nucleus and nucleolar separation occurs. Continued invagination of the nuclear envelope in the region of the interzonal spindle eventually separates the daughter nuclei. A remnant of the interzonal spindle persists in the cytoplasm until cytokinesis.     

Mitosis in the dermatophyteMicrosporum canis as revealed by freeze substitution electron microscopy

Summary Mitosis in the dermatophyteMicrosporum canis was studied by freeze substitution and electron microscopy, and analyzed by three dimensional reconstruction from serial sections of the mitotic nuclei. The interphase nucleus has associated nucleus-associated organelle (NAO) on a portion of the outer surface of the nuclear envelope, subjacent to which there was dense intranuclear material. The NAO divided and separated on the envelope, and a spindle was formed. The spindle was composed mostly of microtubules extended between opposite NAOs. Pairing of kinetochores was observed in the spindle from an early stage of development, when chromosomes were not so condensed, and remained unchanged while chromosome condensation proceeded until metaphase. Before the completion of nuclear division, daughter nuclei were connected by a narrow spindle channel, and then the nucleolus, whose structure underwent minimal change during mitosis, was eliminated into the cytoplasm.     

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.     

ULTRASTRUCTURE AND DIFFERENTIATION IN CLADOPHORA GLOMERATA. I. CELL DIVISION

Cladophora glomerata is a coenocytic, fresh-water green alga in which mitosis and cytokinesis occur independently. The mitotic spindle is centric, closed, and develops from two half-spindles which form from amorphous but well-defined MTOCs at each pole. The nucleolus is only partially dispersed during mitosis and structured kinetochores are evident on the chromosomes. Anaphase separation of chromosomes is asynchronous and results from spindle elongation plus shortening of the chromosome-to-pole distance. Neither a phycoplast nor a phragmoplast is present during cytokinesis. Microtubules are associated with the septum but whether they participate actively in its ingrowth is not clear. Two types of vesicles are associated with the growing septum. The membrane at its leading edge is thicker and more densely stained than elsewhere. The ultrastructure of nuclear and cell division in C. glomerata is sufficiently different from the data on other green algae that conclusions about phylogeny must await further study, especially of other coenocytic green algae.     

Cell division in Hymenomonas carterae (Braarud et Fagerland) Braarud (Prymnesiophyceae)

The cell division cycle of Hymenomonas carterae (Braarud et Fagerland) Braarud was investigated at the ultrastructural level. DNA synthesis and cytokinesis occurred during the 8-hour dark period. All organelles, including the flagellar bases were replicated prior to nuclear division. Prophase consisted of a clustering of the chromosomes into distinct groups and the disappearance of the nucleolus. During metaphase there was complete dissociation of the nuclear envelope resulting in the formation of an open spindle containing no major organelles. The metaphase plate formed at right angles to an imaginary line joining the two pairs of flagellar bases. Elongation of the cell and separation of the chromosomes occurred at anaphase. During early telophase the nuclear envelope veformed and was closely associated with the chromosome masses, resulting in the nuclear possessing convoluted profiles. Telophase was characterized by complete break down of spindle fibres, rounding off of the nuclear profiles, reappearance of the nucleolus, emergence of the flagella and the final separation of the two daughter cells.     

Mitosis and cytokinesis inTribonema regulare (Tribophyceae,Chrysophyta)

Summary The ultrastructure of mitosis and cytokinesis of the uninucleateTribonema regulare has been investigated by employing transmission electron microscopy. Prophase is characterized by settlement of a pair of centrioles at the presumptive poles of the spindle, metaphase by equatorial bulging of the nucleus, anaphase by non-synchronous separation of the chromosomes, and telophase by a persistent, strongly elongated, interzonal spindle. Throughout mitosis, at each pole dictyosomes are associated with the polar gaps of the nuclear envelope that otherwise remains intact. Cytokinesis does not immediately follow mitosis; from the static images it can be concluded that it is necessary for the daughter nuclei to approach each other before cytokinesis is initiated by complete division of the protoplast via plasma membrane cleavage. Afterwards, a ring of cell wall material is deposited close near the lateral wall in the plane of protoplast separation followed by a simultaneous or centripetal development of a single integral partitioning septum. Once the septum is completed, the cylindrical portion of the H-shaped segment is manufactured. The phylogenetic position ofTribonema amongst those algae, which may have evolved from unicells into filaments, is discussed.     

Kinetochore and microtubules in two members of Chlorophyceae,Cladophora fracta and Spirogyra majuscula

Evidence is presented for the existence of a localised kinetochore with stratified fine structure in Cladophora and in Spirogyra. In the latter, there is the possibility of two kinetochores on the longer chromosomes. There is no evidence for a diffuse kinetochore. The nucleolus persists during mitosis in Cladophora on the nucleolar organising chromosomes, the granular material being lost from it very largely during metaphase and anaphase but the fibrillar material remaining. The persistent nucleolar material at metaphase and anaphase in Spirogyra is not attached to the nucleolar organising chromosomes but accumulates around all the chromosomes and chromatids, the microtubules of the spindle at anaphase passing through and possibly attaching to this nucleolar material and possibly assisting in the movement of the chromatids which are embedded within it.     

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.     

小报春与岩生报春种间杂交亲和性研究

以报春花属报春花组的小报春(Primula forbesii)和指叶报春组的岩生报春(Primula saxatilis)为亲本,对种间杂交的结实性及花粉管行为进行了观察。结果表明:小报春与岩生报春种间杂交表现为不亲和,正反交组合正常结实率为0,但正交、反交组合花粉在柱头表面的萌发和花粉管伸长过程有明显差异,小报春花粉授粉4h后可以在岩生报春柱头表面萌发,但花粉管伸长的速度明显比对照组[岩生报春(P)×岩生报春(T)、岩生报春(T)×岩生报春(P)]慢,并且花粉管生长弯曲,授粉192h后花粉管仍未到达子房;岩生报春的花粉可以在小报春柱头上正常萌发,授粉48h后花粉管到达子房;4个对照授粉组合均分别于24h(短花柱为母本)、48h(长花柱为母本)时完成受精过程。研究表明,岩生报春×小报春杂交存在受精前障碍,小报春×岩生报春杂交亲和性较好,并可通过幼胚拯救的方法获得组间杂种后代;花柱长度可能是影响种间杂交结实能力的因素之一。     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Formation of the nuclear envelope during mitotic anaphase inSpirogyra

The formation of the nuclear envelope in the mitosis ofSpirogyra was studied with an electron microscope. The nuclear envelope was disrupted around the spindle equator in the metaphase. Many small vesicles were observed in the metaphase spindle. These vesicles surrounded the masses of chromosomes and nucleolar substance in the early anaphase, and they fused with each other to form daughter nuclear envelopes during the early anaphase. The formation of new envelopes from small vesicles at such an early mitotic anaphase is reported here for the first time. The possible origin of these vesicles is also discussed.     

An immunofluorescence study of mitosis in a mite-pathogen,Neozygites sp. (Zygomycetes: Entomophthorales)

Summary Mitosis in a mite-pathogenic species ofNeozygites (Zygomycetes: Entomophthorales) was investigated by indirect immunofluorescence microscopy using an antibody against -tubulin for visualization of microtubules (MTs). DAPI and rhodamine-conjugated phalloidin were used to stain chromatin and actin, respectively. Salient features of mitosis inNeozygites sp. are (1) a strong tendency for mitotic synchrony in any given cell, (2) conical protrusions at the poles of metaphase and anaphase nuclei revealed by actin staining, (3) absence of astral and other cytoplasmic MTs, (4) a spindle that occupies most of the nuclear volume at metaphase, (5) a spindle that remains symmetrical throughout most of mitosis, (6) kinetochore MTs that shorten during anaphase A, (7) a central spindle that elongates during anaphase B, pushing the daughter nuclei into the cell apices, and (8) interpolar MTs that continue to elongate even after separation of the daughter nuclei. Cortical cytoplasmic MTs are present in a few interphasic and post-cytokinetic cells. The data presented show thatNeozygites possesses features unique to this genus and support the erection of theNeozygitaceae as a separate family in theEntomophthorales.Abbreviations DAPI 4,6-diamidino-2-phenylindole - MT microtubule - SPB spindle pole body     

Unusual pattern of mitosis in the free-living flagellateDimastigella mimosa (Kinetoplastida)

Summary The three-dimensional ultrastructural organization of the mitotic apparatus ofDimastigella mimosa was studied by computer-aided, serial-section reconstruction. The nuclear envelope remains intact during nuclear division. During mitosis, chromosomes do not condense, whereas intranuclear microtubules are found in close association with six pairs of kinetochores. No discrete microtubule-organizing centers, except kinetochore pairs, could be found within the nucleus. The intranuclear microtubules form six separate bundles oriented at different angles to each other. Each bundle contains up to 8 tightly packed microtubules which push the daughter kinetochores apart. At late anaphase only, midzones of these bundles align along an extended interzonal spindle within the narrow isthmus between segregating progeny nuclei. The nuclear division inD. mimosa can be described as closed intranuclear mitosis with acentric and separate microtubular bundles and weakly condensed chromosomes.Abbreviation MTOC microtubule-organizing center     

Ultrastructural studies of the cell cycle in a multicentriolar form ofBracteacoccus minor (Chlorophyceae,Chlorellales)

Summary The ultrastructure of mitosis and cytokinesis is studied in the typical and a multicentriolar form of the multinucleate green algaBracteacoccus minor (Chodat) Petrovà. These processes are essentially identical in both forms, and are similar to those in other uni- and multinucleate chlorellalean algae. The mitotic spindle is closed and centric, and a fragmentary perinuclear envelope is present. In multinuclear cells mitosis is synchronous and may occur at the same time as cytokinesis. Cleavage is simultaneous and centrifugal, starting near the nucleus-associated centrioles and apparently mediated by phycoplast microtubules of the trochoplast type. Flagellated wall-less spores are usually formed. In the typical form ofB. minor, each interphase nucleus is associated with two mature centrioles (= one set) which function as centrosomal markers. At the onset of mitosis these centrioles duplicate and segregate and eventually establish the two poles of the spindle, where polar fenestrae develop in the nuclear envelope. In the multicentriolar form, however, each interphase nucleus generally is associated with two or three sets of centrioles. Consequently, during mitosis each half-spindle is associated with two or three sets. These centrioles are not necessarily all associated with the fenestrae at the spindle poles, but one or more sets are frequently associated with the nuclear membrane, more or less remote from the nuclear poles. However, the spindle in this multicentriolar form remains essentially bipolar. Cleavage generally results in zoospores with two, four or six flagella. The behaviour of the extra centrioles during the cell cycle and their possible relationship with centrosomes are discussed.     

CELL DIVISION IN CHLAMYDOMONAS MOEWUSII1

Cell division in Chlamydomonas moewusii is described. The cells become immobile with flagellar abscission prior to mitosis. The basal bodies migrate toward the nucleus and become intimately associated with the nuclear membrane which is devoid, of ribosomes where adjacent to the basal bodies. The basal bodies replicate at preprophase. The nucleolus fragments at this stage. By prophase the basal body pairs have migrated, to the nuclear poles. Spindle fibers become prominent in the nucleus. The nuclear membrane does not fragment. The nucleus assumes a crescent-form by metaphase. Polar fenestrae are absent. Kinetochores appear at anaphase. An interzonal spindle elongates as the chromosomes move to the nuclear poles. Daughter nuclei become abscised by an ingrowth of nuclear membrane, leaving behind a separated, degenerating interzonal spindle. Ribosomes reappear on the outer nuclear membrane at late telophase. Nucleoli reform early in cytokinesis. The cleavage furrow, associated microtubules, and endoplasmic reticulum comprise the phycoplast. Cytokinesis proceeds rapidly after the completion of telophase. The basal body-nucleus relationship becomes reorganized into the typical interphase condition late in cytokinesis. Specific and predictable organelle rearrangements during mitosis have been described. Cell division in C. moewusii is compared with other algae, especially C. reinhardi.     

MITOSIS AND CLAMP FORMATION IN THE FUNGUS PORIA MONTICOLA

Nuclear division in P. monticola is in general similar to mitosis in higher organisms. Synchronous division of the nuclei in the dikaryon progresses with clamp development. Mitosis begins with the movement of the centriolar plaques into and under the forming clamp. The pull of the centriolar plaque on the attached nucleolus forms a long strand of nucleolar material. Chromosomes now appear as dense granules at the end of the nucleus proximal to the clamp. At this time the nucleolus moves adjacent to the centriolar plaque and contracted chromosomes. The nuclear membrane at least partially disintegrates, and the nucleolus is released into the cytoplasm where it may persist through telophase. A faintly staining spindle is often observed, and it produces a “double bridge” effect in separating chromatin. Somatic chromosomes are attached together forming strings that appear double and at least partially separated before metaphase.