The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Summary Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.     

THE ULTRASTRUCTURE OF CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE (PLASMODIOPHOROMYCETE)

Vegetative nuclear divisions in cystosoral Plasmodia from the shoot system of Sorosphaera veronicae Schroeter were studied with standard transmission electron microscopy. Each metaphase nucleus forms a cruciform configuration as the persistent nucleolus elongates perpendicularly to chromatin aligned on the equatorial plate. The nuclear envelope remains intact during metaphase and anaphase. Each spindle pole consists of a fenestrated nuclear envelope with an exteriorly situated centriole and closely associated endoplasmic reticulum. Intranuclear membranous vesicles occur within metaphase and anaphase nuclei and are closely associated with chromatin and nuclear envelope. Microtubules pass from centrioles into the nucleus and are also attached to chromatin at kinetochores.     

Mitosis of Micronuclei During Division and Regeneration in the Ciliate Stentor coeruleus1

Stages of mitosis of the micronuclei of Stentor coeruleus were described as seen by transmission electron microscopy. Cells in division and those regenerating new oral membranelles were studied. Microtubules were found in early prophase in the karyoplasm and interspersed between the condensing chromatin. A monaxial intranuclear spindle is formed by early metaphase, with kinetochore microtubule attachment sites on the chromosomes. The spindle elongates, separating the daughter nuclei at anaphase. A new nuclear envelope, consisting of two unit membranes, begins to form at late anaphase. Small segments of membrane found in the space between the newly forming and the old micronuclear envelopes appear to fuse to form the new nuclear envelope. No ultrastructural differences were found in the mitotic nuclei of cells in division or regeneration.     

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

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

Low and high voltage electron microscopy of mitosis and cytokinesis in maize roots

The structure and distribution of cytoplasmic membranes during mitosis and cytokinesis in maize root tip meristematic cells was investigated by low and high voltage electron microscopy. The electron opacity of the nuclear envelope and endoplasmic reticulum (ER) was enhanced by staining the tissue in a mixture of zinc iodide and osmium tetroxide. Thin sections show the nuclear envelope to disassemble at prophase and become indistinguishable from the surrounding ER and polar aggregations of ER. In thick sections under the high voltage electron microscope the spindle is seen to be surrounded by a mass of tubular (TER) and cisternal (CER) endoplasmic reticulum derived from both the nuclear envelope and ER, which persists through metaphase and anaphase. At anaphase strands of TER traverse the spindle between the arms of the chromosomes. The octagonal nuclear pore complexes disappear by metaphase, but irregular-shaped pores persist in the membranes during mitosis. It is suggested that these form a template for pore-complex reformation during telophase. Phragmoplast formation is preceded by an aggregation of TER across the spindle at anaphase. Evidence is presented to suggest that the formation of the desmotubule of a plasmodesma is by the squeezing of a strand of endoplasmic reticulum between the vesicles of the cell plate.Abbreviations CER cisternal endoplasmic reticulum - ER endoplasmic reticulum - HVEM high voltage electron microscope - TER tubular endoplasmic reticulum - ZIO zinc iodide/osmium tetroxide     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum II. Metaphase I—Telophase I

Summary The three-dimensional structure of the spindle pole body (SPB) and meiotic spindle during early metaphase I through telophase I inPuccinia malvacearum is analyzed ultrastructurally from serial sections. During early metaphase I the spindle rotates from the perpendicular to a position oblique to the longitudinal axis and parallel to the sagittal plane of the cell. Tubular cisternae are present within the spindle at this stage. The half middle piece (MP) subtends a collateral disc (co-disc) which is inserted eccentrically within each SPB. The SPB, co-disc and half MP at opposite poles are in mirror image. During the transition from early metaphase I to full metaphase I, the spindle orients parallel to the lateral wall of the promycelium and the half MPs are lost. The co-discs partially detach from each discoid SPB and maintain this relation until the end of interphase I. Co-discs become further differentiated as they attach to the subtending sheath-like extension of the nuclear envelope previously occupied by the half MPs. Microvesicles within the nucleoplasm are specific to mid metaphase I. A metaphase plate is absent. The 14 bivalents, which are directly connected to each polar SPB by 2 to 3 kinetochore MTs, are spread over nearly the entire length of the central spindle. The first anaphasic movement involves asynchronous shortening of the kinetochore MTs while the second consists of extensive pole-to-pole elongation. Astral MTs first appear at early metaphase I and become most numerous at anaphase I. An intact nuclear envelope constricts against the central spindle at either end of the interzonal region. Concurrently, centripetal growth of the nuclear envelope under each SPB results in their gradual externalization by the end of telophase I. The sibling nuclei are cut off by constriction of the nuclear envelope at either end of the interzonal region. These meiotic stages inP. malvacearum are compared with those in other basidiomycetes and ascomycetes.     

CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

Associations between membranes and microtubules during mitosis and cytokinesis in caulonema tip cells of the mossFunaria hygrometrica

Summary The interphase nucleus in theFunaria caulonema tip cells is associated with many non-cortical microtubules (Mts). In prophase, the cortical Mts disappear in the nuclear region; in contrast to moss leaflets, a preprophase band of Mts is not formed in the caulonema. The Mts of the early spindle are associated with the fragments of the nuclear envelope. Remnants of the nucleolus remain in the form of granular bodies till interphase. The metaphase chromosomes have distinct kinetochores; the kinetochore Mts are intermingled with non-kinetochore Mts running closely along the chromatin. Each kinetochore is associated with an ER cisterna. ER cisternae also accompany the spindle fibers in metaphase and anaphase. In telophase, Golgi vesicles accumulate in the periphery of the developing cell plate where no Mts are found. The reorientation of the cell plate into an oblique position can be inhibited by colchicine. It is concluded that the ER participates in controlling the Mt system, perhaps via calcium ions (membrane-bound calcium ions have been visualized by staining with chlorotetracycline) but that, on the other hand, the Mt system also influences the distribution of the ER. The occurrence and function of the preprophase band of Mts is discussed.     

Mitosis in the protostelidPlanoprotostelium aurantium

Summary Mitosis in the protostelidPlanoprotostelium aurantium Olive andStoianovich is characterized by an open, centric spindle. The nuclear envelope breaks down prior to metaphase, begins to reform during late anaphase, and is complete by telophase. Centrioles are present at the poles throughout mitosis and are devoid of rootlet microtubules from metaphase to late anaphase. Chromosomes are small and numerous and are attached to single kinetochore microtubules during metaphase and early anaphase. Chromosome separation takes place by a presumed shortening of the chromosome to pole spindle followed by a lengthening of the interzonal spindle. Mitosis inP. aurantium is similar to that of certain other protostelid amoebae and to myxomycete amoebae, but it is considerably different from that of dictyostelid amoebae. The phylogenetic significance of this is discussed.This research represents part of a Ph.D. dissertation presented to the University of North Carolina.     

The ultrastructure of mitosis inIsochrysis galbana parke (Prymnesiophyceae)

Summary Mitosis and cytokinesis have been studied in the flagellate algaIsochrysis galbana Parke (Prymnesiophyceae). Nuclear division is preceded by replication of the flagella and haptonema, the Golgi body and the chloroplast; fission in the chloroplast occurs in the region of the pyrenoid. During prophase, spindle microtubules radiating from two ill-defined poles are formed. The nuclear envelope breaks down and the chromatin condenses. At metaphase the spindle is fully developed, some pole-to-pole microtubules passing through the well-defined chromatin plate, others terminating at it. No kinetochores or individual chromosomes were observed. By late metaphase, many Golgi-derived vesicles may be seen against the two poleward faces of the metaphase plate. During anaphase, the two daughter masses of chromatin move towards the poles. In early telophase, the nuclear envelope of each daughter nucleus is complete only on the side towards the adjacent chloroplast, remaining open on the interzonal side. However, during telophase each nucleus becomes reorientated so that it lies lateral to the long axis of the spindle and with its open side towards the chloroplasts. By late telophase, each new nuclear envelope is complete and confluence with the adjacent chloroplast ER established.Cytokinesis and subsequent segregation of the daughter cells are effected by the dilation of Golgi- and ER-derived vesicles in the interzonal region. No microtubular structures are involved. Comparisons with the results from other studies of mitosis in members of thePrymnesiophyceae show that they all have a number of features in common, but that there are differences in detail between species.     

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.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.     

Spindle membranes and calcium sequestration during meiosis ofDysdercus intermedius (Heteroptera)

The fate of intracellular membranes stained by the osmium ferricyanide (OsFeCN) procedure was followed from premeiotic interphase to interkinesis inDysdercus intermedius. During diakinesis the centrioles forming primary cilia attach temporarily with their proximal ends to the nuclear envelope which is stretched from pole to pole. Breakdown of the nuclear envelope is preceded by deep indentations with microtubules from growing asters. Vesicles of smooth endoplasmic reticulum which accumulate gradually in the course of prophase contribute to the ensheathment of the chromosomes with membranes. When the nuclear envelope breaks down, the polar parts of the formerly perinuclear membranes follow the ingrowth of the spindle microtubules towards the cell equator where the seven bivalents are arranged in a circle with the X₁X₂ sex chromosomes in the centre. The metaphase I spindle thus contains longitudinally oriented membranes between the poles, membranous envelopes around all chromosomes and radial connections from the autosomes to the sex chromosomes in the centre. At anaphase the homologues leave their common sheath and a microtubular stembody surrounded by membranes appears between the receding dyads. In the interkinetic nucleus the gonosomes are separated from the autosomes by a common membranous sheath which may be instrumental in their joint assignment to only one pole in the second meiotic division. Calcium sequestering sites visualized by oxalate precipitation are the Golgi lamellae and vesicles derived from them that surround the whole spindle body.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.     

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.     

Meiosis,spindle pole body cycle,and taxonomy of the heterobasidiomycetePachnocybe ferruginea

Meiosis and the meiotic spindle pole body cycle were studied electron microscopically in basidia of the heterobasidiomycetePachnocybe ferruginea. Spindle pole body splitting in prometaphase I and II, and intermeiotic and postmeiotic duplication were investigated in particular detail. During prophase, the spindle pole body consists of two three-layered discs connected by a middle piece. At late prophase I and again in prometaphase II, the discs contact the nuclear envelope. Then, the nuclear membrane at the contact area is separated from the non-contacted part of the nuclear envelope and finally disappears. Each disc nests into the nuclear opening of the otherwise intact nuclear envelope. The disc remains in the gap and generates a half spindle. At late metaphase I, a co-disc develops eccentrically within the parent disc. The co-disc detaches from the parent disc during interphase I and becomes one of the metaphase II spindle pole bodies. Co-discs are absent during the second division. A cap of endoplasmic reticulum encloses each disc during prophase I through anaphase I. In the second meiotic division, the caps covering the spindle pole bodies of one nucleus of the pair, are developed from the neighbouring nucleus. Spindle pole bodies ofP. ferruginea are similar to those of the rusts, and especially to those ofEocronartium muscicola andHelicobasidium mompa. Part 73 of the series Studies inHeterobasidiomycetes.     

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.