Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Demonstration of different patterns of microtubule organization in Physarum polycephalum myxamoebae and plasmodia using immunofluorescence microscopy

We have used anti-tubulin antibodies and immunofluorescence microscopy to determine the overall distribution of microtubules during interphase and mitosis in both the myxamoebae and plasmodia of the slime mold Physarum polycephalum. We have paralleled these observations with electron microscopy of the same stages. The myxamoebae possess a network of cytoplasmic microtubules whilst the coenocytic plasmodium does not possess any cytoplasmic microtubules--at either interphase or mitosis. In plasmodia microtubules are, however, elaborated by an intranuclear microtubule organizing centre (MTOC) during prophase of mitosis and these microtubules proceed to form part of the mitotic spindle. There is little difference in the overall distribution and arrangement of microtubules during division of either the myxamoebal or plasmodial nuclei. These findings are discussed in relation to the synthesis of tubulin during the plasmodial cell cycle and the rearrangements of the nuclear envelope during mitosis.     

Dynamics of the profiles of plasmodial veins ofPhysarum polycephalum during the pulsation cycle

Summary The cross-sectional profiles of isolated veins ofPhysarum polycephalum plasmodia, winded round agar rods, were investigated by analysis of time-lapse films. The whole profile follows the same principal contraction-relaxation rhythm, but slight desynchronization and amplitude differences are found around the contour. As a result, the profile periodically changes its shape in the course of pulsation cycles, its lateral slopes becoming more convex in the expanded state than during contraction. Factors responsible for such geometrical deformations and their bearing for morphometrical studies of the motory activity of plasmodium are discussed.Study supported by the Research Project II.1 of the Polish Academy of Science.     

CRUCIFORM NUCLEAR DIVISION IN WORONINA PYTHII (PLASMODIOPHOROMYCETES)

Somatic nuclear divisions in sporangiogenous plasmodia of Woronina pythii Goldie-Smith were studied with transmission electron microscopy. During metaphase, each nucleus formed a cruciform configuration as chromatin became aligned at the equatorial plate perpendicular to the persistent nucleolus. Except for polar fenestrations, the original nuclear envelope remained intact throughout the mitotic division. Intranuclear membranous vesicles appeared to bleb off the inner membrane of the original nuclear envelope, adhered to the surfaces of the separating chromatin, and eventually formed new daughter nuclear envelope within the original nuclear envelope. During the first 24 hr of vegetative plasmodial growth, each telophase nucleus exhibited an obvious constriction of the original nuclear envelope in the interzonal region. Similar constrictions were not evident in telophase nuclei found in 24–36-hr-old plasmodia. This variation in the ultrastructural morphology of cruciform division appears to be related to the age and size of each sporangiogenous plasmodium, and is the first to be documented within this group of fungal pathogens.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.     

Cell cycle events during the development of the silk glands in the mulberry silkworm<Emphasis Type="Italic"> Bombyx mori</Emphasis>

Silk glands of the mulberry silkworm Bombyx mori are long and paired structures originating from the labial region and are anatomically and physiologically divided into three major compartments, the anterior, middle and posterior silk glands. The silk gland morphogenesis is complete by 8 days post egg laying. Extensive growth of silk glands during the larval stages is due to increase in tissue mass and not cell number. The cells in a completely formed silk gland pursue an endoreplicative cell cycle, and the genome undergoes multiple rounds of replication without mitosis or nuclear division. The expression patterns of cyclin B (mitotic cyclin) and cyclin E (G1 cyclin, essential for G1/S transition in both mitotic and endoreplicative cell cycles) in the course of silk gland development revealed that mitotic cell divisions take place only in the apex of the growing silk gland. However, the persistence of another mitotic focus in the middle silk gland even when the growing apex has moved well past this zone suggested the continued operation of mitosis for a while in this restricted region. The lack of cyclin B expression and abundance of cyclin E in the rest of the areas confirmed an alternation of the G1 and S phases of the cell cycle without an intervening mitotic phase. No expression of cyclin B was noticed anywhere in the silk glands after stage 25 of embryogenesis, indicating a complete switch over to the endomitotic mode of the cell cycle. The onset of expression of various genes encoding different silk proteins correlated with the onset of endomitotic events.Edited by D. Tautz     

Information processing for the organization of chemotactic behavior ofPhysarum polycephalum studied by micro-thermography

Summary The spatial and temporal pattern of oscillating temperatures on the cell surface of a plasmodial strand ofPhysarum polycephalum was measured with a sensitive thermal image camera. The longitudinal tension of the strand was studied simultaneously. In the absence of chemical stimulation, the phases of the temperature oscillation observed at various portions of the strand were entrained with almost coincidental phase. The temperature and tension oscillation were synchronized, although the phase difference between them was occasionally changed. With local chemical stimulation, the phase of the temperature oscillation advanced in the portion to which the plasmodium would be induced to migrate. The phases between temperature and tension oscillations then became constant. The mechanism by which the plasmodium processes local information of chemical stimulus to global information for the migration is discussed.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.     

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.     

Syngamy and plasmodium formation in the MyxomyceteDidymium iridis

Summary Cell and nuclear behavior during syngamy in the MyxomyceteDidymium iridis and the development of zygotes into plasmodia by both synchronous mitoses and by coalescence with other zygotes and plasmodia are described. Various aspects of cell and nuclear behavior are discussed in relation to the induction of syngamy and the trigger mechanisms responsible for switching the course of development from one pathway to another.     

Growth of Large Plasmodia of the Myxomycete Physarum polycephalum

A method has been developed for growing Physarum polycephalum plasmodia that are 8 to 10 times larger than those obtained in the petri dish cultures used by Nygaard, Guttes, and Rusch. In the large-scale procedure, plasmodia were grown in metal trays on a membrane supported by filter paper on stainless-steel screen. Plasmodia were started from a ring of inoculum to allow inward and outward migration and were incubated on a rocker so that nutrient medium would flow back and forth, wetting the undersurface of the plasmodium. Rocker and petri dish cultures had similar growth characteristics: (i) the interphase time between mitoses I and II and between II and III was about 8 hr; (ii) ribonucleic acid and protein increased essentially logarithmically throughout the cell cycle; and (iii) deoxyribonucleic acid increased only during early interphase and it doubled in approximately 3 hr after each mitosis. Rocker cultures were not as nearly synchronous as petri dish cultures and had a range in metaphase time (at mitosis III) within individual plasmodia of 15 to 45 min, as compared with 5 to 10 min in petri dish cultures.     

The basal body-root complex ofChlamydomonas reinhardtii during mitosis

Summary The results of this work clarify several structural and temporal aspects of biogenesis of the basal body-root complex inChlamydomonas reinhardtii. The two phases of basal body development (probasal body assembly and conversion of probasal body into mature basal body) occur at identical mitotic stages in successive mitoses during multiple fission, which indicates a tight coupling between basal body development and the mitotic cycle. The two steps of basal body development are separated from one another in time,i.e. immature probasal bodies originate during an interval lasting ca. 5 min between mid-metaphase and early telophase, but mature after a quasi-dormant period only during early prophase of the next mitotic round. The duration of the dormant period depends on the interval between two mitoses: during synchronized vegetative growth there is an interval of ca. 20 h (interphase growth) between two rounds of multiple fissions, but only a maximum interval of 1.5 h between the successive mitoses of one round of multiple fissions.The microtubular root system, which is bisected at the same time as the basal body apparatus in a plane perpendicular to the distal connecting fiber during prophase, and whose roots seem to be reduced in length, starts duplication at early metaphase with the successive origin of two short bud-like partner roots just opposite the remnants. These initial roots elongate during subsequent phases by unilateral and radial growth from the basal bodies and along the cell's periphery, but exactly where they terminate is not known. The two-stranded roots opposite each other appear to be again connected as early as anaphase.The striation pattern of the distal connecting fiber is lost during early prophase thus indicating a partial breakdown of the fiber.Dedicated to Prof. Dr. C.-G. Arnold (Erlangen) on the occasion of his 60th birthday.     

Modulation of cellular rhythm and photoavoidance by oscillatory irradiation in the Physarum plasmodium

We studied responses of cellular rhythm and light-induced movement to periodic irradiation in a unicellular amoeboid organism, the Physarum plasmodium. The intrinsic frequency of the contraction rhythm, which is based on biochemical oscillations, became synchronized with the frequency of periodic irradiation with light when both frequencies were close enough. In order to study the role of the synchronization in light-induced movement, periodic irradiation was applied to only part of the plasmodium. The rate of avoidance of light was modulated in the frequency band in which the synchronization occurred. The synchronization property of the contraction oscillation underlies the regulation of tactic movement in plasmodium.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

Entamoeba histolytica: cell cycle and nuclear division

The cell cycle of Entamoeba histolytica, the duration of its phases, and the details of the nuclear division stages are described in this paper. Trophozoites from clone L-6, strain HM1:IMSS, were synchronized by colchicine. Synchrony was observed immediately after treatment and cultures remained synchronous for at least three replicative cycles with synchrony indexes between 13 and 15 hr. The stages of nuclear division were studied by light and electron microscopy. Four stages of the nuclear division were defined: prophase, early anaphase, late anaphase, and telophase. No metaphase stage was observed by light or electron microscopy. One of the first events in the nuclear division was the presence of a bud close to the juxtanuclear body, which grew to a daughter nucleus. The karyosome and the nuclear membrane remained throughout the mitotic process. Bundles of intranuclear microtubules were observed forming a "V" from the center of the nucleus to one of the poles, and associated with them, 12 to 16 chromosomes-like structures appeared. The results of these studies strongly suggest that division of E. histolytica involved a pleuromitotic process which is carried out in about 120 min.