Cleavage Vesicles Research Articles

Ultrastructure of the Zoosporangia of Albugo ipomoeae-panduratae as Revealed by Conventional Chemical Fixation and High Pressure Freezing Followed by Freeze Substitution

Both conventional chemical fixation and high pressure freezing followed by freeze substitution (HPF/FS) were used to prepare zoosporangia of the oomycete Albugo ipomoeae-panduratae inside infected host leaves for study with transmission electron microscopy. Both fixations gave good preservation of ultrastructural details and data from the two sample types were highly complementary. However, HPF/FS gave better overall specimen contrast and superior preservation of microtubules, basal bodies and curved vacuoles closely associated with basal bodies. The basal body-associated vacuoles appear to represent cleavage vesicles involved in zoospore formation. Although HPF/FS did result in the rupture of some vacuoles and the extraction of lipid bodies, these problems did not interfere with our study. Overall zoosporangium morphology was similar to that reported previously for A. candida. Each zoosporangium was multinucleate and contained numerous mitochondria, lipid bodies, a variety of large and small vacoules/vesicles, and conspicuous arrays consisting of parallel strands of rough endoplasmic reticulum. Golgi cisternae and a pair of basal bodies were closely associated with each nucleus.

The Ultrastructural Development of Sporangiospores in Multispored Sporangia of Zygorhynchus heterogamus with a Hypothesis for Sporangial Wall Dissolution

The ultrastructural development of multispored sporangia in Zygorhynchus heterogamus was found to be similar to the established general model for sporangiosporogenesis in multispored sporangia. Shortly after the initiation of a sporangial swelling, numerous cleavage vesicles appeared within the highly vacuolate precleavage sporangium. These vesicles appeared to fuse, forming an interconnecting network of cleavage furrows which ramified throughout the young sporangium. Cleavage furrows, lined with electron-opaque granules, isolated the numerous spore protoplasts. Synchronous with spore cleavage, the electron-opaque granules fused and formed the exterior spore wall layer. As the spore cytoplasm condensed, the spore walls matured with a corresponding reduction of electron-translucent material between the spores. With autolysis of the columellar cytoplasm, numerous vesicles appeared within the columella and fused with its plasma membrane. At the same time, endogenous degradation of the sporangial wall occurred, followed by the release of mature spores. Laser scanning confocal fluorescence microscopy revealed that the sporangial wall contained chitin, but neither the columellar nor the sporangiospore walls did.

Comparative ultrastructural ontogeny of zoosporangia of Zygorhizidium affluens and Z. planktonicum, chytrid parasites of the diatom Asterionella formosa

The fine-structural development of the small, 5–10 m, operculate zoosporangia of the monocentric chytrids Zygorhizidium affluens and Z. planktonicum , growing as epibionts on the planktonic diatom Asterionella formosa , is compared. In both species, following encystment and germination, the germ tube grows over the surface of the diatom frutule and penetrates the host cells by squeezing between the upper and lower girdle lamellae. As the cyst enlarges to form a sporangium, the single lipid globule breaks up and disperses, an operculum differentiates and the nucleus divides. In both species, cleavage of the cytoplasm into zoospore initials is mainly achieved by the infurrowing of the plasmamembrane, although there may be some contribution from internal cleavage vesicles. Flagellar axoneme profiles are not observed in the sporangia until cytoplasmic cleavage is well advanced. Specialized zoospore organelles, such as the rumposome fibrillar vesicles and endoplasmic reticulum-delimited ‘ribosome core’, do not form until the later stages of zoospore differentiation. The taxonomic and functional implications of these observations are discussed.

Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes

Rapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.

Ultrastructure of zoosporogenesis in Phytophthora cinnamomi

Zoospore formation in Phytophthora cinnamomi has been studied by electron microscopy in conjunction with immunogold labelling of selected cellular elements. Zoosporogenesis involves the compartmentalization of the mature multinucleate sporangium into a number of biflagellate, uninucleate zoospores. Before the process is initiated, the pyriform nuclei of the sporangium are regularly distributed throughout the cytoplasm. Several cellular elements, including basal bodies and dictyosomes, are found exclusively or predominantly near the narrow poles of nuclei. A second order of organization involves the nuclei of the sporangial cortex whose narrow poles point toward the wall. These patterns are critical to the sequence of changes during cleavage and in establishing the dorso-ventral axis of the zoospore. Zoosporogenesis is initiated by a cold shock, and within 20 min large clusters of cleavage vesicles appear near each narrow nuclear pole and elsewhere in the cytoplasm. Monoclonal antibody Cpw-1 labels material closely associated with the membrane of the cleavage vesicles and also binds to nearby dictyosomes which are proposed as the source of the vesicles. Between 20–35 min after induction, the large clusters of cleavage vesicles near the narrow poles of nuclei in the cortex disperse to form a plane of vesicles parallel to the wall. These vesicles are closely aligned with microtubules that fan out from the basal bodies. Between 25–50 min after induction, clusters in the sporangial interior disperse, and their vesicles align in planes that link with the plane of vesicles in the cortex. The planes of vesicles surround polyhedral domains of cytoplasm that contain one nucleus. Fusion of these vesicles brings about compartmentalization of the sporangium. The antigen labelled by Cpw-1 is closely associated with the plasma membrane of the newly formed zoospore initials. This is the first time that monoclonal antibodies have been used to identify the source and fate of cleavage vesicles in a fungal sporangium, and the results of this study suggest that aspects of the cleavage process in other Phytophthora species may need to be reassessed.

A complementary experimental study of cell division in the dinoflagellate Prorocentrum micans

Cell division in Prorocentrum micans was studied with TEM, SEM and with fluorescence microscope techniques to visualize cytoskeletal structures. The cleavage furrow consists of the plasma membrane and two valvar vesicles. Its leading edge is lined with actin filaments. The completion of cytokinesis (like in some other dinoflagellates) is inhibited by extremely low doses of cytochalasin D. Under the influence of this drug cleavage vesicles fuse into large cleavage cavities. New valvae are formed, however, with an irregular, warty surface, caused by a surplus of membrane material and/or lack of stretch and possibly influenced by a predetermined pattern of the cell surface. At the end of mitosis individual or small groups of chromosomes are surrounded by the nuclear envelope.

Ultrastructure of Zoosporogenesis in the Endoparasite Olpidiopsis varians

The fine structure of zoosporogenesis in the endoparasite Olpidiopsis varians is described and compared to other oomycetous fungi. The wall of the parasite zoosporangium is composed of an inner granular to fibrous material and an outer layer of electron opaque material. Prior to cytoplasmic cleavage approximately 8–12% of the zoosporangial nuclei disintegrate. Cleavage furrows are produced throughout the parasite cytoplasm at a time when large, centrally situated vacuoles are present. The fusion of cleavage vesicles delimits the parasite zoospore initials which possess a central spherical nucleus, spherical mitochondria, dense-body vacuoles, mastigoneme packets, two kinetosomes with attached microtubular rootlets, a single kinetosome-associated organelle, and lipid bodies. The exit tube of the zoosporangium grows through the host wall and zoospores are released to the outside.

Ultrastructure of Zoosporogenesis in the EndoparasiteOlpidiopsis Varians

The fine structure of zoosporogenesis in the endoparasite Olpidiopsis varians is described and compared to other oomycetous fungi. The wall of the parasite zoosporangium is composed of an inner granular to fibrous material and an outer layer of electron opaque material. Prior to cytoplasmic cleavage approximately 8–12% of the zoosporangial nuclei disintegrate. Cleavage furrows are produced throughout the parasite cytoplasm at a time when large, centrally situated vacuoles are present. The fusion of cleavage vesicles delimits the parasite zoospore initials which possess a central spherical nucleus, spherical mitochondria, dense-body vacuoles, mastigoneme packets, two kinetosomes with attached microtubular rootlets, a single kinetosome-associated organelle, and lipid bodies. The exit tube of the zoosporangium grows through the host wall and zoospores are released to the outside.

Pearl millet downy mildew (Sclerospora graminicola): Zoosporogenesis

Cleavage of the zoosporangial cytoplasm ofSclerospora graminicola, the causal agent of pearl millet downy mildew, is by means of the fusion of cleavage vesicles and vesicles containing the extruded axoneme with the cell membrane. This type of zoosporogenesis linksS. graminicola to other Peronosporalean species, and is very similar to that seen for all uniflagellate species examined to date, while it separates it from species of theSaprolegniales where zoosporogenesis is brought about by the expansion of the central vacuole, or where the plasmalemma alone is used. The origin of the cleavage vesicles appears to be from the dictyosomes and not from the finger-print bodies which are rapidly formed in large numbers after axoneme formation and after the cleavage. vesicles have started to appear in the cytoplasm.

The Golgi apparatus, zoosporogenesis, and development of the zoospore discharge apparatus of Chytridium confervae

C. confervae is a eucarpic, monocentric chytrid that has been cultured synchronously ( L. P. Gauriloff and M. S. Fuller, 1979, Exp. Mycol. 3: 3–5). In this study we use light and electron microscopy to examine the development of zoospores and the discharge apparatus, focusing on the multiple roles of the Golgi apparatus. The Golgi apparatus produces, in succession, vesicles with electron-opaque cores that may function in cell wall formation, secretory vesicles that form the extracellular lenticular deposit of the discharge apparatus, cleavage vesicles that fuse to form the plasma membranes of the developing zoospores, and vesicles that contain cell coat material for the zoospores. The discharge apparatus consists of the operculum (a circle of sporangial wall delimited from the rest of the wall), the lenticular deposit, and an outer layer found between the lens and the operculum. At discharge, the operculum dehisces, the outer layer ruptures, and the lenticular deposit expands to form a vesicle that constrains the zoospores. The outer layer provides the mechanical connection between the wall and the vesicle. Comparison of discharge apparatus development with other Chytridiomycetes suggests that the order of the developmental steps leading to discharge may be as important to chytrid taxonomy as the steps themselves.

An ultrastructural study of the marine diatom Licmophora hyalina and its parasite Ectrogella perforans. II. Development of the fungus in its host

The thallus of the fungus Ectrogella perforans Petersen inside its host, the diatom Licmophora hyalina Agardh, is surrounded initially by two electron-dense membranes, of which the outer one is the invaginated host plasma membrane and the inner one, the fungal plasma membrane. Later, new membranes are added between these two membranes and the fungal envelope consists of four to six membranes. When the fungal thallus is mature, all the membranes except the fungal plasmalemma break down and it secretes an amorphous wall around itself. This coincides with the breakdown of host organelles followed by death of the host cell. Zoosporogenesis begins after the sporangium becomes multinucleate. A peculiar "multitubular body" is always observed in the multinucleate sporangium. A typical feature of the multinucleate sporangium prior to zoosporogenesis is the presence of a ring of tubular cisternae around the nuclei, electron-dense vesicles, and granular vesicles.The tubular cisternae found around the nuclei move away and act as cleavage cisternae. The cleavage cisternae run perpendicular to the sporangial plasma membrane and delimit the sporangial mass into uninucleate units at the time of zoosporogenesis. Simultaneously, vesicles are pinched off from the Golgi body which act as cleavage vesicles. These cleavage vesicles fuse with each other and form cleavage furrows. The cleavage cisternae fuse with the plasma membrane outside and with the cleavage vesicles inside and thus deepen the cleavage furrows. The sporangial mass is thus divided into zoospore units and the units are connected only by narrow cytoplasmic bridges. The zoospores have their flagella developed already. The structure of primary zoospores, encysted primary zoospores, and encysted secondary zoospores is described here.

Cycle, ultrastructure of a Catenaria (Phycomycetes Blastocladiales) parasite of Crustacea Cyclopoida

The ultrastructural modifications during the life cycle of a Phycomycetes parasite of Cyclops are studied. The cytoplasmic sporangium cleavage is brought about by the fusion of flagellar sheaths and cleavage vesicles. The uniflagellates zoospores are characterized by a nuclear-nuclear cap cluster, a large side-body (single mitochondrion, lipid bodies, granular material) and gamma particles. The fungus belongs to the genus Catenaria and probably to the species C. anguillulae. The ultrastructure of this Catenaria shows that this genus is closely related to Blastocladiales.

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea.

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross-bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.

Ultrastructure of the Termite-Associated Fungus Laboulbeniopsis Termitarius

SUMMARY Ultrastructural features of Laboulbeniopsis termitarius suggest that this termite-associated fungus may belong to ascomycetes. Spores are formed without cleavage vesicles typical of sporangial fungi, abundant epiplasm remains, and septal walls between cells of simple thallus bear a single pore. A single ascus forms from base of terminal sporogeneous cell, but deliquesces by time of spore maturation. Haustoria are not present, although secretory vesicles were noted in two areas of foot cell suggesting mode of attachment. Although many aspects of this fungus suggest relationships with Laboulbeniales, uniascal thallus, one-celled spores, and lack of haustoria exclude Laboulbeniopsis from this taxon. Thaxter (1914, 1920) described a number of fungi from insects which could not be clearly assigned to existing taxonomic groups. He pointed out their unclear taxonomic position and lack of knowledge of their nutritional relationships with insects on which they occur. Two of these monotypic genera, Laboulbeniopsis and Coreomycetopsis, were described and placed by Thaxter (1920) in the limbo genera incertae sedis, and in same paper he suggested with reservation that Laboulbeniopsis might be accommodated in Thaxteriolae with Thaxteriola Speg., Endosporella Thaxt., and possibly Entomocosma Speg. Laboulbeniopsis termitarius and Coreomycetopsis oedipus were not mentioned again for fifty years until Kimbrough and Gouger (1970) reported them from Florida on species of termite genus Reticulitermes. Their light microscopy study added little to what was known about mature thallus. However, many aspects of thallus development, spore formation, and spore liberation and attachment were discussed. Since many morphological details of L. termitarius cannot be discerned with certainty at light microscopic level, we undertook

An ultrastructural study of zoosporogenesis inPythium proliferum de Bary

Zoosporogenesis in the oomycete,Pythium proliferum de Bary initially involves a condensation of cytoplasm at certain hyphal tips and the subsequent enlargement of these hyphal tips to form sporangia. Deposition of a septum at the base of the sporangium and initiation of an apical papilla are followed by cleavage of the sporangial cytoplasm. Packets of presumptive mastigonemes as well as flagella are recognized in the cytoplasm at this time. Subsequently, cleavage vesicles at the periphery of the sporogenic cytoplasm fuse with the plasmalemma thereby emptying their fibrous contents into the space between the sporogenic cytoplasm and the sporangial wall. It is felt that this fibrous material is instrumental in developing the internal pressure necessary within the sporangium to cause discharge of the sporogenic cytoplasm into an evanescent vesicle wherein delimitation of the zoospores is completed. The formation of the spore vesicle from the multilayered apical cap of the papilla is described here for the first time in the Oomycetes and a new term, “vesiculogen”, is suggested for this structure. Aspects of centriole replication within vegetative hyphae, papilla formation, and morphogenesis of various vesicular inclusions are also described in this study.

Ultrastructure of chlamydospores of Phytophthora cinnamomi during development and germination

Thin sections of developing, fully expanded, and germinating chlamydospores of Phytophthora cinnamomi were studied by electron microscopy. Expanding chlamydospores had thin walls and contained nuclei, mitochondria, dictyosomes, rough endoplasmic reticulum, lipid-like bodies, peripheral vesicles, and small vacuoles with dense, spherical inclusions. Small vesicles, about 200 nm in diameter, were occasionally observed underlying the plasmalemma.In fully expanded chlamydospores, structures that are necessary for zoosporogenesis, i.e. exit pores, flagella, and cleavage vesicles, were not present. The vacuoles with dense, spherical inclusions constituted a large proportion of fully expanded chlamydospores and had usually fused to form a large central vacuole. The walls of two-week-old chlamydospores were similar in density to sporangial walls and were non-layered.Chlamydospore germination was direct, i.e. by the production of germ tubes with numerous vesicles at the tips which ruptured through the chlamydospore wall. A germination wall was formed de novo beneath the chlamydospore wall and extended with the germ tube to form the germ tube wall.The distinguishing ultrastructural characteristics between chlamydospores and sporangia, aged sporangia, oogonia, and oospores of Phytophthora are discussed.

Ultrastructure of Zoosporogenesis in Allomyces macrogynus

SUMMARY The formation of zoospores in the aquatic Phycomycete, Allomyces macrogynus, has been studied with the electron microscope. Flagellum formation, zoospore-membrane formation and nuclear-cap formation comprise the sequence of major events that occur within a zoosporangium upon the addition of water to a culture of the organism growing on an agar surface. At least two distinct kinds of cleavage vesicles were found: those that delimit the zoospore and those that delimit the flagellum and nuclear cap. The vesicles that form the zoospore membrane originate from the sporangium membrane while vesicles that form the primary flagellar vesicle and nuclear cap may originate from cisternae of the endoplasmic reticulum.

Ultrastructural Changes Associated with Spore Formation in Sporangia and Sporangiola of Thamnidium elegans Link

Fully formed pre-cleavage sporangia and sporangiola of Thamnidium elegans Link were bounded by a primary wall plus a thick, internal secondary wall layer. In sporangia in late pre-cleavage, Golgi-like cisternae were associated with groups of cytoplasmic vesicles of characteristic size and appearance which were not found in sporangia containing large cleavage vesicles. In both sporangia and sporangiola, protoplast cleavage was effected by enlargement of endogenous cleavage vesicles each containing a lining layer of variable appearance, mutual fusion of cleavage vesicle membranes and fusion of cleavage vesicle membranes with the plasmalemma. Golgi-like cisternae and small vesicular profiles were present in sporangium protoplasts at all stages of cleavage vesicle enlargement. In sporangia, the columella zone was delimited by cleavage vesicles and separated from the sporogenous zone by a fibrillar wall. A similar wall, which sometimes protruded to form a small columella, was formed in sporangiola. Recently delimited spore protoplasts were bounded by plasmalemma membrane derived from cleavage vesicle bounding membrane and sporangium or sporangiolum plasmalemma and surrounded by an investing layer derived from cleavage vesicle lining material. The investing layer at first appeared single, but later two electron opaque profiles were discernible. The spore wall was formed between the investing layer and the plasmalemma. Walls of sporangia and sporangiola which contained fully formed spores consisted of the primary layers only.

ULTRASTRUCTURE OF ZOIDOGENESIS IN UNILOCULAR ZOIDOCYSTS OF SEVERAL BROWN ALGAE1

SUMMARYSeveral different stages of the development of unilocular zoidocysts of small brown algae—Elachista fucicola, Hecatonema streblonematoides, Pylaiella littoralis—are observed by electron microscopy.1. A slow growing phase is seen, during which nuclei and pheoplasts become associated by pairs and divide together, vacuoles and physodes are excreted through the plasmalemma, and Golgi bodies liberate vesicles with fibrillar material identical to the growing cell wall fibers. Mitochondria and Golgi bodies are concentrated under the very sinuous plasmalemma.2. A very short spatial reorganization phase follows, during which organelles disperse between the nuclei‐pheoplast pairs, cleavage vesicles appear, and flagella start developing. New pyrenoids form de novo.3. The latter phase is followed by a longer maturation phase. Cleavage vesicles fuse and separate zoids grow as flagella. Mastigonemes formed in the endoplasmic reticulum are finally found in vesicles of a special Golgi body at the base of the anterior flagellum. They are liberated in parallel rows at the base of the already developed flagella by these Golgi's vesicles, and attach, on the flagella by an unknown process. Excretion of a mucilaginous substance takes place as the stigmas form de novo.4. The ripe, swollen zoidocysts burst open, liberating the whole gelatinous mass. Naked zoids swim and settle on a substrate, retracting their flagella before excreting a new cell wall.

Electron microscopy of the sporangium of Phytophthora capsici

This paper reports results of a study on the ultrastructural cytology of sporangia of Phytophthora capsici Leonian with emphasis on flagellum formation, general sporangial structure during zoospore cleavage, and the presence, structure, and transition of cytoplasmic organelles and inclusions during these processes.Non-cleaving sporangial cytoplasm contains a high concentration of ribosomes, mitochondria, vacuoles, lipid inclusions, and endoplasmic reticular cisternae scattered throughout the cytoplasm. Nuclei in mature sporangia are located at the periphery of the cell, with their narrow poles aligned towards the plasma membrane. The apical papilla measures 4–5 × 10 μ, and is initiated as a fibrous third layer under the two-layered cell wall several microns from the apex. The outer wall layer surrounds the papilla while the inner wall narrows and disappears near the crown. The basal septal plug is a combination of the inner wall layer and slime substances.One of the first structural changes in the cytoplasm during zoospore cleavage is the genesis of the flagellar apparatus. Paired centrioles next to the narrow poles of the nuclei elongate to form kinetosomes which extend through the cytoplasm toward receptive axonemal vesicles. Axonemes then form in the axonemal vesicles. The terminal plate and its prisms account for the appearance of the axoneme when it forms above the terminal plate in the axonemal vesicle. The axonemal cylinder has a typical 9 + 2 morphology and the axonemal sheath is continuous with the axonemal vesicle tonoplast. The nucleus is an integral part of the flagellar apparatus and appears to be connected to the kinetid (axoneme + kinetosome) base via microtubules. Golgi dictyosomes occur in the sporangia during all stages of growth and may be responsible for elaboration of needed membranes during zoospore production. Osmiophilic droplets (liposomes), located within vacuoles, are a predominant feature of precleavage cytoplasm. These globules are probably lipid in nature. As cleavage begins, the liposomes become less opaque at the margins, and striations appear, eventually encompassing all or most of the liposomes at the time of cleavage. The liposomes then become less spherical and expand, filling the vacuoles. Electron-transparent regions eventually appear throughout the liposomes and the vacuolar membrane may disappear.Cleavage of the cytoplasm into zoospores occurs by the alignment and fusion of cleavage vesicles around individual nuclei. During this period organelles migrate to these centers. The cleavage vesicles coalesce with each other and the axonemal membranes, eventually becoming the plasma membranes of the daughter zoospores.