The gamma body: A vesicle generating structure

Gamma bodies, which are present in the sporangia and gametangia of Allomyces and in its spores, are interpreted as constituting vesicle generating structures. During spore cleavage the mobilization–decay of the gamma bodies leads to vesicle formation; the vesicles appear to fuse to form the axonemal and plasma membrane of the spore. Vesicle formation by the gamma bodies during spore cleavage can be perturbed by phosphate buffer which leads to the formation of myelin–figure arrays of membranes, or by colchicine and benomyl which give rise to large vacuolar structures after gamma body decay. During the motile period of the spores of Allomyces , mobilization of the gamma bodies leads to vacuole formation and the resulting vacuoles fuse with the plasma membrane of the spore and by this means maintain the osmotic balance of the spore. During spore encystment the gamma body decays and forms vesicles which fuse with the plasma membrane of the cyst; these vesicles appear to be instrumental in chitin wall synthesis.     

Functional necessity of the cytoskeleton during cleavage membrane development and zoosporogenesis in Allomyces macrogynus

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.     

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 effect of colchicine on colony formation in the algae Hydrodictyon,Pediastrum and Sorastrum

Summary Uninucleate, biflagellate zoospores of Hydrodictyon, Pediastrum and Sorastrum, derived from multinucleate parental cells, aggregate and adhere to form distinctively patterned colonies; earlier work has shown that microtubules underlie the plasmalemma of these zoospores and are also conspicuous in the developing horns of aggregating cells of Pediastrum and Sorastrum. Colchicine applied to parental cells inhibited cytoplasmic cleavage and production of uninucleate zoospores. When zoospores were treated with colchicine, their peripheral microtubules disappeared; the spores failed to aggregate in ordered arrays and did not develop horns. The microtubules therefore appear to play an important role in determining the arrangement of cells in developing colonies by affecting the shape of the cells at the time of their aggregation.     

Pearl millet downy mildew (Sclerospora graminicola): Zoosporogenesis

Summary 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.     

Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.     

Differential effects of antimitotic agents on the stability and behavior of cytoplasmic and ciliary microtubules

Summary When sea urchin gastrulae are treated with colchicine or hydrostatic pressure the cytoplasmic microtubules disappear, but the ciliary microtubules which make up the ciliary axoneme (9+2) remain. With calcium-free sea water the cytoplasmic microtubules are reduced in number yet the 9+2 complex in the cilia is unaffected. Furthermore during the administration of any of these agents the cilia continue to beat so that functionally as well as morphologically the ciliary microtubules are normal even though the cytoplasmic microtubules are broken down and their presumed function in development is interrupted.Available evidence indicates that these two types of microtubules appear to be made up of similar subunits. Since there are morphological connections between the microtubules of the ciliary axoneme, and since the ciliary microtubules appear to stain more intensely than the cytoplasmic microtubules, we conclude that the ciliary microtubules are stabilized either by the addition of material or through interactions between adjacent tubules or both.Supported by Grant #5T 1-GM-707 from the National Institutes of Health to ProfessorKeith R.Porter.     

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS : II. The Role of Nucleating Sites in Shape Development

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.     

A light and electron microscopic study of sporulation in the myxomyceteStemonitis virginiensis

Summary The conversion of the plasmodium ofS. virginiensis into sporophores has been examined at both the light and electron microscopic levels. Particular attention has been paid to stalk and columella formation, capillitial formation, nuclear behavior during sporulation and spore formation. Both the stalk and columella are formed within the sporangial initial as intraprotoplasmic secretions. A portion of the capillitium arises directly from the columella while the remainder forms within an anastomosing system of tubular vacuoles. As spore cleavage begins the nuclei within the sporangium begin to divide mitotically. The protoplasmic content of the sporangium is first divided into small protospores which typically contain a single dividing nucleus. Following the completion of mitosis each of these segments cleaves into yet smaller segments which develop into spores. Meiosis occurs in the spores some 12–16 hours after cleavage.     

CHEMICAL AND MORPHOLOGICAL STUDIES OF BACTERIAL SPORE FORMATION : IV. The Development of Spore Refractility

From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers—cortex, coat, and exosporium—in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is ⅓ fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whiten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions.     

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.     

Studies on the effects of vinblastine and colchicine on Trypanosoma gambiense

Vinblastine and colchicine induce the anucleate form of Trypanosoma gambiense. Light microscopic studies indicated that the anucleate form was not always produced as a result of inhibition of nuclear duplication, but was formed as a result of delay or inhibition of separation of the two nuclei after completion of nuclear division. Studies showed that vinblastine and colchicine caused disorder in arrangement of axonemal microtubules of the extracellular flagella and increased formation of both protofilaments and the axoneme composed of protofilaments in trypanosomes. Moreover, treatment with colchicine resulted in disintegration of previously existing pellicular microtubules and formation of cytoplasmic projections that appeared as protrusions from a small part of the surface membrane.     

THE ROLE OF MICROTUBULES IN THE MOVEMENT OF PIGMENT GRANULES IN TELEOST MELANOPHORES

When microtubules in teleost melanophores are disrupted with antimitotic agents, colchicine, high hydrostatic pressure, low temperature, and vinblastine, the alignment and movement of the pigment granules in these cells disappear; during recovery, the return of alignment and movement corresponds in both time and space with the repolymerization of microtubules. Furthermore, analysis of nearest neighbor distances in untreated melanophores reveals that pigment granules are closely associated with microtubules. Other structures such as microfilaments, the endoplasmic reticulum, and the cytoplasmic matrix do not appear to be involved. Thus we conclude that microtubules determine the alignment and are essential for the selective movements of the pigment granules in these cells. Investigations of the mechanism of movement show that microtubules are required for both centrifugal and centripetal migrations and that they do not change in number or location during redistribution of pigment. Our results further indicate that microtubules in melanophores behave as semistable organelles as determined by investigation with colchicine and hydrostatic pressure. These observations and others rule out a push-pull mechanism based on the polymerization and depolymerization of microtubules or one which distinguishes two operationally different sets of microtubules. We propose instead that particles move by sliding along a fixed array of microtubules.     

Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. II. An experimental analysis of their role in development and maintenance of cell shape

To experimentally test the suggestion made in the preceding paper that the microtubules are involved in cell shape development during the formation and differentiation of the primary mesenchyme, we applied to the embryos two types of agents which affect cytoplasmic microtubules: (a) colchicine and hydrostatic pressure, which cause the microtubules to disassemble, and (b) D₂O, which tends to stabilize them. When the first type of agent is applied to sea urchin gastrulae, the development of the primary mesenchyme ceases, the microtubules disappear, and the cells tend to spherulate. With D₂O development also ceases, but the tubules appear "frozen," and the cell asymmetries persist unaltered. These agents appear to block development by primarily interfering with the sequential disassembly and/or reassembly of microtubules into new patterns. The microtubules, therefore, appear to be influential in the development of cell form. On the other hand through a careful analysis of the action of these agents and others on both intra- and extracellular factors, we concluded that the microtubules do rather little for the maintenance of cell shape in differentiated tissues.     

TWO TYPES OF CYTOPLASMIC CLEAVAGE IN THE COENOCYTIC SOIL ALGA PROTOSIPHON BOTRYOIDES (CHLOROPHYCEAE)1

Cytokinesis in the coenocytic green alga Protosiphon botryoides (Kütz.) Klebs was studied with transmission electron microscopy. In vegetative cells, nuclei with associated basal bodies and dictyosomes are scattered throughout the cytoplasm. Mature cells may develop either multinucleate resting spores (coenocysts) or uninucleate zoospores. Cytokinesis may be preceded by contraction of the protoplast due to the disintegration of vacuoles that are present in larger, siphonous cells. The formation of coenocysts in ageing, siphonous cells, is signalled by cleavage of the chloroplast and the development of arrays of phycoplast microtubules in one or more transversely oriented planes through the cell. Nuclei with associated basal apparatuses stay dispersed throughout the cytoplasm; the basal bodies apparently are not involved in organization of the phycoplast. The plasma membrane invaginates, resulting in a centripetal cleavage of the protoplast into two or more multinucleate daughter protoplasts. Simultaneously, wall material is deposited along the outside of the daughter protoplasts by dictyosome-derived vesicles, and finally two or more thick-walled coenocysts are formed. The formation of zoospores, on the other hand, is signalled by clustering of the nuclei in one or more groups depending on the shape of the mother cell. The nuclei become arranged with the associated basal apparatuses facing toward the center of the cluster. Bundles of phycoplast microtubules develop between the nuclei, radiating from the center of a cluster toward the plasma membrane; basal apparatuses or associated structures apparently are involved in organization of the phycoplast. Cleavage furrows grow out centrifugally along these bundles of micro-tubules, fed by dictyosome-derived vesicles. No wall material is deposited. An additional mitotic division occurs during cleavage, and finally numerous uninucleate, wall-less, biflagellate zoospores are formed. The ultrastructural features of the two different types of cytoplasmic cleavage associated with two different types of daughter cells have not previously been reported for chlorophycean algae.     

Desmosome assembly in MDCK epithelial cells does not require the presence of functional microtubules.

Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstrated an apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules.     

ULTRASTRUCTURE OF SPOROGENESIS IN A MOSS,DITRICHUM PALLIDUM. III. SPORE WALL FORMATION

Ultrastructural evidence indicates that marked cytoplasmic polarity occurs during wall and aperture ontogeny in spores of the moss (Musci), Ditrchum pallidum (Hedw.) Hampe. Shortly after cytokinesis, an extensive system of microtubules underlies the entire distal spore surface where exine deposition is initiated. These microtubules appear to be focused on the plastid. The apposition of slips nearly of membrane dimension contributes to the forming exine. As the lamellate exine thickens and extends to the proximal surface, the plastid and associated nucleus migrate to the proximal surface where an elaborate system of microtubules involved in aperture development is generated. The exine gradually loses its stratiform character, becoming homogenous and eventually papillate. At maturity, the spore wall consists of four layers, the outermost perine, the exine, a separating layer, and the intine. The aperture is a complex, localized modification of these layers on the proximal surface. It consists of a pore containing a fibrillar material surrounded by a thin annulus.     

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of γ-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from γ-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

苔藓植物孢子发生的研究进展

苔藓植物孢子发生的过程是一个复杂的形态建成的过程,在此过程中,孢子母细胞经过减数分裂的两次精确的核分裂以及细胞质分裂,形成单倍体的四分孢子,再经孢子壁的发育过程,形成成熟的孢子。本文重要介绍了苔藓植物孢子发生过程中细胞质裂片、质体及核的变化、微管系统及纺锤体、胞质分裂和孢子壁形成过程的特点及其研究进展。