CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

Nuclear Degeneration during Ballistospore Formation of Cronartium asclepiadeum (Uredinales)*

When basidia of Cronartium asclepiadeum (Uredinales) develop basidiospores, nuclei migrate from the basidial cells into the basidiospores. A mitotic nuclear division yields two nuclei in the basidiospore. One of these nuclei degenerates if the basidiospore develops a secondary ballistospore. Several stages in the degeneration of the nuclei can be recognized: (1) Condensation of chromatin, (2) separation of the nucleus into a portion containing the chromatin and a portion containing the nucleoplasm by an invagination of the nuclear envelope, (3) reduction of nuclear volume, (4) enveloping and (5) twisting of nuclear membranes around the degenerating nucleus, (6) homogenization of the chromatin with reduction of the nuclear envelope, and reduction of the enclosing membrane complex from a multilamellar structure to a single membrane layer, and (7) invagination or splitting off of the spiralled membranes. The nuclear behavior during early developmental stages of secondary spore formation is similar to that of budding basidiomycetous yeasts. Basidiomycetes producing ballistospores may possibly have arisen from those whose reproducing phase is yeast-like.     

Origin and behavior of bicentriolar centrosomes in the bryophyteRiella americana

Summary Basal bodies in embryophyte spermatozoids develop from centrosomes which arisede novo in spermatid mother cells (SMC). The centrosomes at SMC spindle poles in those land plants producing biflagellated sperms comprise a coaxial pair of centrioles, a bicentriole (BC). Ultrastructural observations of antheridia of the aquatic liverwortRiella americana indicate that the centrosome is first evident as a dark staining body on the outer surface of the nucleus. Numerous short microtubules (MT) diverge from this body which next separates into two lobes, each with divergent MTs. Within each lobe, a BC differentiates-the cartwheel hub and spokes developing before the triplet MTs. Constituent centrioles of each BC are apposed by their proximal ends and connected only by the central hub. As the BCs migrate toward opposite spindle poles, they appear to be connected by MTs that terminate in granular material partially investing each BC. Each spermatid resulting from SMC division will inherit a bicentriole.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

COLONY DEVELOPMENT IN EUDORINA ELEGANS (CHLOROPHYTA,VOLVOCALES)1

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.     

The ultrastructure of mitosis in myxamoebae and plasmodia of Physarum flavicomum

Electron microscopy of glutaraldehyde-osmium-fixed samples of haploid myxamoebae and diploid plasmodia of the myxomycete Physarum flavicomum Berk. reveal dissimilar spindle apparatus during mitosis in the two cell types. Myxamoebae exhibit an astral type of mitosis with centrioles at the poles and nuclear envelope breakdown during prophase. Plasmodial nuclei lack centrioles at mitosis and have an intranuclear spindle, with nuclear envelope persisting during the entire division. Coated vesicles are noted during prophase and telophase in myxamoebae and their role in spindle formation and dispersion is suggested.     

Mitosis and autospore formation in the green algaKirchneriella lunaris

Summary Asexual reproduction inKirchneriella lunaris involves autospore formation. After an initial mitosis, the curved cell cleaves to a variable extent, and then the nuclei divide again; finally the cytoplasm is partitioned into four around each nucleus. Rudimentary centrioles appear prior to the first mitosis; centriole complexes then become associated with a developing sheath of extranuclear microtubules at prophase; fenestrae appear at the poles through which both microtubules and centrioles migrate, preceding intranuclear spindle formation. The nucleus meanwhile is enveloped by a perinuclear layer of endoplasmic reticulum which is also interposed between the golgi body and nuclear envelope. Chromosome separation is accompanied by considerable spindle elongation. Finally the reforming nuclear envelope excludes both centriole complex and interzonal spindle apparatus from daughter nuclei. Cleavage is preceded by i) nuclear movement to the cell center, ii) movement of centriole complexes around daughter nuclei until they are opposite one another, and iii) the concurrent formation of a system of transverse microtubules extending across the cell. Other microtubules encircle the cell predicting the cleavage plane. A septum then appears amongst these cytokinetic microtubules, possibly derived from the plasmalemma; it extends across the cell too, through the cleaving peripheral chloroplast. Secondary mitoses follow (as above) during which this septum may be partially resorbed. Finally this septum is reformed, if necessary, and two other septa appear (as above) to quadripartition the cell. Mitotic and cytokinetic structures in this algae are briefly compared with some others.     

EFFECTS OF MICROTUBULE INHIBITORS ON PRONUCLEAR MIGRATION AND EMBRYOGENESIS IN FUCUS DISTICHUS(PHAEOPHYTA)1

Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed.     

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.     

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.     

The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro

Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.     

Mitosis in flat PTK2-human hybrid cells

The fusion of G0 human fibroblasts with PTK₂ (Potorous tridactylis) cells resulted in the production of hybrid heterokaryotic cells which remained flat in cell division. These cells permitted studies of mitosis in living hybrid cells without the need for fixation and staining. The breakdown of nuclear envelopes during prophase in a hybrid heterokaryotic cell correlated with the onset of premature chromosome condensation (PCC) in other nuclei in the same cell. Nuclear morphology and autoradiography demonstrated that the nuclei exhibiting PCC were from the human parent cells. Observation of multinucleated PTK₂-human hybrids in the later stages of mitosis showed that these cells normally produced three daughters instead of the usual two. Electron microscopic examination of dividing hybrid cells showed that the number of daughter cells was not related to the number of centrioles. Hybrid cells normally were found to contain many centriolar duplexes although not all of these structures were associated with active poles in mitosis. Cells with as many as six centriolar duplexes were found in mitosis. The configuration of the chromosomes in metaphase was found to be a more accurate indication of the number of daughters produced by a single division than the number of centrioles. Chromosome elimination in hybrid cells could also be visualized in PTK₂-human hybrids. Lagging chromosomes were commonly observed during mitosis and were often trapped in the constricting midbody.     

Ultrastructure of Draparnaldia glomerata (Chaetophorales, Chlorophyceae) II. Mitosis and cytokinesis

The mitosis and cytokinesis of Draparnaldia glomerata as examined here by transmission electron microscopy are in many aspects similar to those described earlier for other chaetophoralean algae. The standard chaetophoralean model of the mechanism of mitosis/cytokinesis is described in detail. Characteristic in this pattern is the movement of sets of centrioles towards the nuclear poles followed by a proliferation of extranuclear microtubules at prophase, the (partial) fusion of centrioles with the spindle poles at metaphase and anaphase, the simultaneous separation of chromosomes apparently caused by both spindle elongation and shortening of the chromosomal microtubules at anaphase, the expulsion of the centrioles by daughter nuclei and finally the non–persistent spindle at telophase. Cytokinesis takes place by formation of a cell plate associated with phycoplast microtubules. The possible function of the phycoplast in cytokinesis in Draparnaldia is 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.     

Closed spindle nuclear division in the plasmodial phase of the acellular slime moldEchinostelium minutum

Summary Mitosis in the plasmodium ofEchinostelium minutum is intranuclear (closed spindle) and centrioles are not present at the spindle poles. The nuclear envelope remains essentially intact throughout mitosis with polar fenestrae appearing in anaphase and persisting through telophase. During anaphase there is a shortening in the distance of the chromosomes to the poles followed by a further separation of the poles. Zippering of microtubules may be the basis for these two anaphasic movements. During telophase the polar MTOCs are extruded into the cytoplasm through the polar fenestrae prior to reconstitution of the nuclear envelope. It is proposed that during sporulation such MTOCs are responsible for the differentiation of the centrioles which subsequently persist in the myxamoebal phase of this species.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Ultrastructural changes during sporangium formation and zoospore differentiation in Blastocladiella Emersonii

Samples from synchronized cultures of Blastocladiella emersonii were examined by electron microscopy from the late log phase to the completion of zoospore differentiation. Log-phase plants contain the usual cytoplasmic organelles but also have an unusual system of large tubules ca. 45 mμ diam that ramify in organized bundles throughout the protoplast. After induction, zoosporangium differentiation requires a 2-hr period in which the nuclei divide, a cross wall forms to separate the basal rhizoid region, and an apical papilla is produced. Nuclear division in B. emersonii is intranuclear with a typical microtubular spindle apparatus and paired, unequal, extranuclear centrioles at each pole. The papilla is formed by a process of localized cell wall breakdown and deposition of the papilla material by secretory granules. Differentiation of zoospores begins when one of the two centrioles associated with each nucleus elongates to form a basal body. The flagella fibers arise from the basal body and elongate into an expanding vesicle formed by the fusion of small secondary vesicles. The cleavage planes are formed by fusion of vesicles similar to those associated with flagellum initiation. When cleavage is complete, each sporangium contains ca. 250–260 uninucleate spore units with their flagella lying in the cleavage planes. Probable fusion of mitochondria to produce the single mitochondrion of the zoospore occurs after cleavage; the mitochondrion does not take its position around the basal body and rootlets until just before zoospore release. The ribosomal nuclear cap is organized and enclosed by a membrane formed through fusion of many small vesicles during a short period near the end of differentiation.     

Electron and immunofluorescence microscopy on the fertilization ofFucus distichus (Fucales,Phaeophyceae)

Summary Processes of fertilization and zygote development inFucus distichus were studied by indirect immunofluorescence microscopy using anti- tubulin antibody and electron microscopy. Just after plasmogamy, sperm aster formation occurs during migration of a sperm nucleus toward an egg nucleus at the center of cytoplasm. Only sparse microtubules (MTs) exist around the egg nucleus. The sperm aster can be observed till karyogamy, but afterwards vanishes. Accompanying sperm aster formation, cortical MTs which are reticulately arranged develop further in the zygotes. In 4 h-old zygotes, characteristic structures which are composed of fine granular masses and consist of intermixed dense and lighter staining areas appear around the nucleus. These structures cannot be detected with anti- tubulin immunofluorescence microscopy. The two centrioles derived from the sperm separate and migrate to both poles. In 4 h-and 8 h-old zygotes, there are no defined MT foci around the zygote nucleus and MTs radiate from the circumference of it. In 12 h-old zygotes, each centriole has migrated to the poles and derivative centrioles are generated. The fine granular masses also migrate to both poles and finally disappear accompanying the appearance of numerous MTs radiating from the poles. Therefore, two distinct MT foci appear from 12 h onwards. Progressive stages of nuclear division were also examined with electron and immunofluorescence microscopy in 16 h-old zygotes. The sperm chloroplast with an eyespot and the sperm mitochondria with an intercristal tubular structure, which are distinctive from those of egg, can be detected after plasmogamy and karyogamy. The sperm chloroplast is still present in 16 h-old zygotes.     

Chromosome cycles of the basidiomycete Cyathus stercoreus (Schw.) De Toni

Summary Chromosome cycles of the basidiomycete Cyathus stercoreus (meiosis and mitosis) are described. The fusion of two nuclei of compatible mating type takes place in the developing basidium at the end of telophase of the presynaptic mitosis. Synapsis follows immediately after nuclear fusion. During synapsis the chromosomes elongate, facilitating pairing. Meiosis and mitosis are essentially similar to those processes in higher organisms. Details of divisional stages are described and illustrated with photomicrographs. The presence of centrioles and spindles is demonstrated. The presence of quadrivalents as well as secondary associations of like chromosomes suggests that Cyathus stercoreus may be a tetraploid species.     

NUCLEAR BEHAVIOR IN THE VEGETATIVE HYPHAE OF CERATOCYSTIS FAGACEARUM

Vegetative nuclear division in Ceratocystis fagacearum (Bretz) Hunt was found to differ from classical mitosis in that: (1) division always occurs perpendicular to the longitudinal axis of the cell, (2) anaphase movement is unilateral and unsynchronized, (3) a spindle occurs only between separating chromatids. Interphase and prophase nuclei and nucleoli are morphologically similar to those in higher plants. At metaphase the associated chromosomes form a bar of chromatin and lie against the hyphal wall. Spindle fibers appear between separating chromatids, perhaps pushing them apart. When nuclear division is complete the nuclei become attenuated and migrate. Vegetative nuclear division in C. fagacearum may be an evolutionary form of classical mitosis.     

Colony formation and inversion in the green algaEudorina elegans

Summary During development of daughter coenobia in the volvocalean algaEudorina a rapid synchronized series of mitotic divisions and cytokineses gives rise to a slightly cup-shaped, patterned array of 16 or 32 cells, the plakea; the nuclei and centrioles of each cell lying at the concave face and the plastids at the convex face. Each cell is connected to its neighbours by cytoplasmic bridges. All cells within a plakea simultaneously elongate and enlarge their nuclear poles; while remaining interconnected by the cytoplasmic bridges at their plastid poles. The result is inversion of the developing coenobia so that the nuclei and centrioles come to lie on the convex, outer surface. Inversion is inhibited by colchicine and cytochalasin B. Both lengthening of the cells and expansion of their nuclear end is apparently mediated by microtubules. Striations on the plasmalemma encircling the bridges are thought to stablize the membrane at these sites during inversion.