Interactions between microtubules and the nuclear envelope during mitosis in a fern

Summary The relationships between microtubules and the nuclear membrane have been studied during the cell division cycle in adventitious roots ofDryopteris filixmas. At prophase, the equatorial band of microtubules hitherto only reported for higher plant cells is present in the cytoplasm. The occurrence of the band is correlated with a striking change in the shape of the nucleus. Microtubules leave the peripheral cytoplasmic band in groups and are found to partially encircle the nucleus. At the same time pole-to-pole fibres also lie close to the nuclear envelope. A specific function for some of these fibres is proposed, and the results are discussed in the light of previous work on the cytoplasmic features of prophase. The behaviour of the nuclear membrane during mitosis and its reformation around the daughter nuclei are described. These results are related to the concept of microtubule organizing centres.     

Nuclear envelope radiating microtubules in plant cells during interphase mitosis transition

Summary The microtubule distribution during the transition from interphase to the mitotic phase was examined at ultrastructural level in large highly vacuolated cells ofNautilocalyx lynchii and in small non-vacuolated cells ofPisum sativum. Both cell types contain, besides preprophase bands and perinuclear microtubules, also microtubules radiating from the nucleus into the transvacuolar cytoplasmic strands and cytoplasm respectively.This microtubule array appears to be nucleated by the cell's nuclear envelope (NE) or NE-surrounding cytoplasm.It is hypothesized that the microtubules radiating from the nucleusinitially play a role in the mobilization of the nucleus whilelater on a stabilized part of this array anchors the nucleus in the plane of cell division, and thus forms a cytoskeletal link between nucleus and division site.Our results are discussed in the light of previous work on cytoplasmic behaviour during interphase-mitosis transition in highly vacuolated plant cells.     

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.Abbreviations EGTA ethylene glycol bis-(-aminoethyl ether), N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - MF microfilament - MT microtubule - PEG polyethylene glycol - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution     

Microtubule organization during zoosporogenesis inAllomyces macrogynus

Summary The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamino-2-phenylindole - dH₂O deionized water - DMSO dimethyl sulfoxide - DS dilute salts solution - G/5 0.1% glucose medium - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOC microtubule-organizing center - PBS phosphate buffered saline - PCM pericentriolar matrix - TEM transmission electron microscopy - VELM videoenhanced light microscopy     

The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae

Summary In uninucleate cells, cytokinesis follows karyokinesis, thereby reestablishing a specific nucleus-to-cytoplasm ratio. In multinucleate cells, cytokinesis is absent or infrequent; no plasmalemma boundary defines the cytoplasmic territory of an individual nucleus. Several genera of large multinucleate green algae were examined with epifluorescence light microscopy to determine whether the patterns of cytoplasmic organization establish nuclear cytoplasmic domains. Randomly spaced nuclei, singular mitotic events and cytoplasmic streaming characterize the organization of two genera,Derbesia andBryopsis (Caulerpales). The cells ofValonia, Valoniopsis, Boergesenia, Ventricaria (Siphonocladales), andHydrodictyon (Chlorococcales) display regularly spaced nuclei which undergo synchronous divisions in a stationary cytoplasm. In the cytoplasm of genera with regularly spaced nuclei, microtubules radiate from all nuclei in late telophase-early interphase. These internuclear microtubule arrays are not found in algal genera with randomly spaced nuclei. It is hypothesized that these microtubule arrays play a role in establishing the cytoplasmic domain of each nucleus in genera with regularly spaced nuclei. Loss of microtubule arrays during the events of mitosis correlated positively with the increasing randomization of nuclear patterns in algae grown in microtubule inhibitors. Cytoplasmic domains were maintained when cells were grown in the same media in the dark. This suggests that, after a round of division, regular nuclear spacing in certain multinucleate algae is reestablished by internuclear microtubule arrays, which are not, however, required to maintain spacing during interphase.Dedicated to the memory of Professor Oswald Kiermayer     

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.     

γ-Tubulin and microtubule organization during microsporogenesis in Ginkgo biloba

This is the first report on -tubulin and microtubule arrays during microsporogenesis in a gymnosperm. Meiosis in Ginkgo biloba is polyplastidic, as is typical of the spermatophyte clade, and microtubule arrays are organized at various sites during meiosis and cytokinesis. In early prophase, a cluster of -tubulin globules occurs in the central cytoplasm adjacent to the off-center nucleus. These globules diminish in size and spread over the surface of the nucleus. A system of microtubules focused on the -tubulin forms a reticulate pattern in the cytoplasm. As the nucleus migrates to the center of the microsporocyte, -tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci of -tubulin give rise to a multipolar prophase spindle. By metaphase I, the spindle has matured into a distinctly bipolar structure with pointed poles. In both first and second meiosis, -tubulin becomes distributed throughout the metaphase spindles, but becomes distinctly polar again in anaphase. In telophase I, -tubulin moves from polar regions to the proximal surface of chromosome groups/nuclei where interzonal microtubules are organized. No cell wall is deposited and the interzonal microtubules embrace a plate of organelles between the two nuclear cytoplasmic domains (NCDs) of the dyad. Following second meiosis, phragmoplasts that form between sister and non-sister nuclei fuse to form a complex six-sided structure that directs simultaneous cytokinesis. -Tubulin becomes associated with nuclei after both meiotic divisions and is especially conspicuous in the distal hemisphere of each young microspore where an unusual encircling system of cortical microtubules develops.     

Actin-endoplasmic reticulum complexes inDrosera

In epidermal cells ofDrosera tentacles that have been preserved for ultrastructural analysis through high pressure freeze fixation and freeze substitution we describe the frequent occurrence of microfilament (MF)-endoplasmic reticulum (ER) complexes. These are found throughout the cytoplasm where they are observed in close association with the plasmalemma (PL), the tonoplast, nuclei, mitochondria, chloroplasts, and microbodies. The MF component of the complexes is identified as actin based on immunogold labelling with actin antibodies. The actin-ER complexes are prominent in the cortical cytoplasm. In this region a network of predominantly tubular ER occupies an intermediary position in which it associates closely with both the PL and the actin MFs. We suggest that the ER, especially those elements adjacent to the PL in the cortical cytoplasm, stabilizes the actin MFs and provides the necessary anchor against which the forces for cytoplasmic streaming are generated.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FS freeze substitution - HPF high pressure freezing - MF microfilaments - MT microtubules - PL plasmalemma     

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.     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

The hyphae ofUromyces appendiculatus within the leaf tissue after high pressure freezing and freeze substitution

Summary The fine structure of the intercellular dikaryotic hyphae of the biotrophic fungusUromyces appendiculatus was studied. High pressure freezing and freeze substitution were used to achieve a closer approximation of the native state than with conventional fixation and dehydration techniques. In addition to organelles previously described in rust fungi, heavily decorated multivesicular bodies (star bodies) were found close to the nuclei. Two types of tubular-vesicular complexes were distributed randomly within the cytoplasm of the hyphae. Furthermore, a more or less pronounced brush-like fibrillar layer on the hyphal walls was detected. The possibility that the latter two structures are correlated with the biotrophic phase of this fungus is discussed.Abbreviations TVC tubular-vesicular complex - MVB multivesicular body - M mitochondrion - N nucleus - NP nuclear pore - S septum - MT microtubule     

Perinuclear microtubules in postnatal rat heart

Cytoplasmic microtubules can be divided into two subpopulations: 1) those adjacent to the nucleus (perinuclear), and 2) those distributed between the myofilament bundles (nonperinuclear). Previous observations (Cartwright and Goldstein, '83) indicate total cytoplasmic microtubule numeric density increases to a maximum at 5-9 days and decreases to the steady value of the adult muscle. We have examined the numeric density (mean numbers of microtubule profiles per micron2 cross-sectional area) of the perinuclear subpopulation and compared it to the numeric density of the total cytoplasmic microtubule population in postnatally developing rat papillary muscle ages 1, 3, 5, 9, 21, and 42 days, and adult. The perinuclear region was defined as the area around the nucleus which extends to the 0.273 micron from the nuclear envelope. The density of perinuclear microtubules did not change with postnatal development. Our study suggests that perinuclear microtubules are a separate and relatively stable subpopulation of the total population of cytoplasmic microtubules and may serve a function different from that of the more variable nonperinuclear microtubules.     

Cell division and nuclear movement in the saccoderm desmidNetrium interruptus

Summary During anaphase in thisNetrium, the reforming daughter nuclei hardly pause at the poles before they elongate and rapidly and smoothly move along the daughter cells in one of the grooves in the chloroplast. Ahead of each nucleus is a pointed mass of cytoplasm that is distinctly striated; straight, mobile strands of cytoplasm emanate from this region ahead of the nucleus. When the nucleus reaches the large vacuole that divides the two chloroplasts, it steadily slides over to the chloroplast surface distal to the cleavage furrow. It then stops moving and slowly expands into the normal interphase morphology.Under the electron microscope, the chromosome-to-pole distance does not decrease much during anaphase (i.e., anaphase A is minimal) and so the half spindles remain about the same length by telophase. The poles of the open spindle are initially broad and contain typical spindle microtubules (MTs). These persist intact during anaphase and become focused upon a discrete Organizing Centre as the daughter nuclei reform. These MTs become a cone-shaped array that creates the pointed cytoplasmic mass ahead of the moving nucleus in live cells. Thus, this placoderm desmid behaves very likeClosterium during division and shows the lack of anaphase A, and the transformation of the telophase spindle into a MT-based motility system, now characteristic of many members of the Zygnematales.Abbreviations MT microtubule - MTOC microtubule organizing centre Dedicated to the memory of Professor Oswald Kiermayer     

IMMUNOFLUORESCENT LOCALIZATION OF MICROTUBULES THROUGHOUT THE CELL CYCLE IN THE GREEN ALGA MOUGEOTIA (ZYGNEMATACEAE)

Indirect immunofluorescence microscopy was used to survey the three-dimensional distribution of microtubules throughout the cell cycle in the green alga Mougeotia. The network of microtubules present in the cortex of the cells at interphase gradually disappeared before mitosis. A band of cortical microtubules reminiscent of the preprophase band of higher plants surrounded the nuclei of some preprophase cells undergoing cortical microtubule disassembly. Longitudinally oriented bundles of microtubules appeared at the future spindle poles on either side of the nuclei in prophase. These bundles disappeared gradually as the spindle microtubule arrays formed. New spindles had broad poles but these became quite pointed before anaphase. Interzonal microtubules appearing at anaphase persisted until the end of nuclear migration, by which time they were concentrated into narrow bundles on either side of the centripetally forming crosswalls. During decondensation of the chromosomes and early nuclear migration, the spindle poles persisted as sites of microtubule concentration. New arrays of microtubules radiated from these microtubule centers into the cytoplasm ahead of the migrating nuclei. After cytokinesis, reinstatement of cortical microtubules was best observed in regions of the cells remote from the nuclei and associated microtubules. In contrast to higher plants, the first detectable cortical microtubules were short and already oriented transverse to the long axes of the cells.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The development of nuclei and cytoplasmic microtubules was studied in the maturing spermatids of the grasshopper, Acrida lata, fixed with glutaraldehyde-potassium bichromate-osmium tetroxide and embedded in epoxy Epon-resin. Utilization of microkaryosomes for the formation of paracrystalline nucleoprotein is suggested by the fact that they are no longer visible in the advanced spermatid nuclei showing the paracrystalline structure. The cytoplasmic microtubules approximately 220 Å in diameter develop in close association with a linear material similar in density to the nuclear envelope. Only a single layer of the double-layered nuclear envelope is visible during the development of microtubules. Although cytoplasmic microtubules are assumed to have several physiological functions, such apparatus seem to be related to the polymerization of nucleoproteins as well, since the depolymerization of nucleoproteins occurs simultaneously along with the disappearance of cytoplasmic microtubules.     

Nuclear migration in a nud mutant of Aspergillus nidulans is inhibited in the presence of a quantitatively normal population of cytoplasmic microtubules

Nuclear migration was studied in germinating conidia of a temperature-sensitive mutant of the fungus Aspergillus nidulans. At the restrictive temperature motility was demonstrably impaired because significantly fewer nuclei migrated into the germ tube relative to a population of similarly sized germlings grown at the permissive temperature. Further comparison of these populations showed that the mutant was leaky in that an increasing number of nuclei migrated as the total nuclear content increased in each germling. The restrictive temperature also induced elevated mitotic asynchrony and increased numbers of nuclei per germling. Serial section-based reconstruction of the microtubules in a freeze-substituted germling showed that they were not attached to the nucleus-associated organelles, were approximately parallel to the long axis of the germ tube, and seemed to be randomly distributed between the central and peripheral cytoplasm. Five germlings from each temperature were selected for quantitative analysis of cytoplasmic microtubules. All 10 germlings had typical nuclear migration phenotypes. No significant temperature-related difference in microtubule density was found. We conclude that inhibition of nuclear migration in the mutant is the effect of some defect other than the failure of cytoplasmic microtubules to assemble to their normal population density. We also suggest that nuclear motility is not dependent on mitosis-related microtubules.     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

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.