Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

四膜虫S1有性生殖的电镜观察Ⅱ.接合区的形态与配子核的交换

四膜虫接合膜上的小孔是两接合体细胞质相连的通道。配子核形成后,按合膜由于增生而出现装有细胞器等的囊状折叠,并可脱离下来而落入另一细胞中,这可能是胞质交流的又一途径。配子核的交换不是从膜上原有小孔通过的,而是在溶酶体等作用下、使膜破裂,由核后方的微管推动进入对侧细胞中。 本文记述了四膜虫S1有性生殖过程中接合区的形态和配子核的交换。     

Spatio-temporal reorganization of intracytoplasmic microtubules is associated with nuclear selection and differentiation during the developmental process in the ciliateTetrahymena thermophila

Summary Indirect immunofluorescence has revealed various intracytoplasmic microtubular structures, which are transiently polymerized in specific subcellular locations during the developmental process of conjugation in the ciliateTetrahymena thermophila. These structures include: (1) micronuclear spindles, (2) perimicronuclear microtubules, (3) microtubular baskets surrounding migrating pronuclei, and (4) microtubules interconnecting the pronuclei with the conjugants' junctional zone. Furthermore, a peripheral network of intracytoplasmic microtubules related to the cell cortex is present in both vegetative cells and in conjugants. Comparative observations made on cells undergoing normal conjugation and defective conjugation (occurring either spontaneously or induced by taxol) has revealed some rules governing the pattern of deployment of conjugation-specific microtubules. The presence of perinuclear microtubular arrays during early postmeiotic stages of development is strictly limited to more anteriorly located nuclei which includes the selected haploid nucleus that further divides to form the stationary and migratory pronuclei. These perinuclear microtubules may be involved in the positional control of nuclear fates leading to effective nuclear selection. Microtubular bundles associated with pronuclei and connecting the junctional zone are only formed in the presence of functional pronuclei, and may be involved in the guidance of pronuclei leading to their fusion. The mechanism of cytoplasmic control of nuclear differentiation of derivatives of the zygotic nucleus appear to be associated with a coordinate action of two microtubular arrays: spindle microtubules of the second postzygotic division and the peripheral intracytoplasmic network of microtubules, leading to a proper subcortical positioning of the postzygotic nuclei at opposite poles of the cell.Abbreviations MTs Microtubules     

Ultrastructure of the Fusion Area during Conjugation in the Suctorian Heliphrya erhardi (Rieder) Matthes*

SYNOPSIS. The suctorian Heliophrya erhardi (Rieder) Matthes is attached to the substrate by the flattened ventral side of the cell body. The dorsal is covered by a pellicle composed of 3 unit membranes. Below the pellicle is a 0.4–0.8-μm thick epiplasm composed of 6–8-nm thick fibrils. Microtubules form a network beneath the epiplasm. The epipalsm is penetrated by tube-like pellicular pits, which are lined by the cell membrane and end beneath the epiplasm in a saccule-like enlargement. During conjugation, 2 neighboring organisms form cytoplasmic processes which come into contact and fuse, thus forming a cytoplasmic bridge between the 2 cells. Around the bridge the pellicles of both organisms fuse, and the partners become united by a continuous common membrane system. Across the entire conjugation bridge the 2 fused epiplasms form a septum. Tube-like structures can be seen lying partly in the epiplasmic septum and partly in the adjacent cytoplasm. These structures are open at both ends and represent remnants of the pellicular pits. No trace of the original pellicular membranes can be found at the fusion area within the epiplasmic septum. The cytoplasm of the conjugation partners is separated only by the fused epiplasms forming the epiplasmic septum.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype.

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time.     

Morphology,Conjugation, and Postconjugational Reorganization of Dileptus tirjakovae n. sp. (Ciliophora,Haptoria)

ABSTRACT. We studied the morphology, conjugation, and postconjugational reorganization of a new haptorid ciliate, Dileptus tirjakovae n. sp., using conventional methods. Dileptus tirjakovae is characterized by two abutting, globular macronuclear nodules and scattered brush kinetids. Conjugation is similar to that in congeners, that is, it is temporary, heteropolar, and the partners unite bulge‐to‐bulge with the proboscis. Some peculiarities occur in the nuclear processes: there are two synkaryon divisions producing four synkaryon derivatives, of which two become macronuclear anlagen, one becomes the micronucleus, and one degenerates. Unlike spathidiids, D. tirjakovae shows massive changes in body shape and ciliary pattern before, during, and after conjugation: early and late conjugants as well as early exconjugants resemble Spathidium, while mid‐conjugants resemble Enchelyodon. These data give support to the hypothesis that spathidiids evolved from a Dileptus‐like ancestor by reduction of the proboscis. Dileptus tirjakovae exconjugants differ from vegetative cells by their smaller size, stouter body, shorter proboscis, and by the lower number of ciliary rows, suggesting one or several postconjugation divisions. Although 83% of the exconjugants have the vegetative nuclear pattern, some strongly deviating specimens occur and might be mistaken for distinct species, especially because exconjugants are less than half as long as vegetative cells.     

A cytogenetic study of development in mechanically disrupted pairs of Tetrahymena thermophila

We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1-3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0-4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. The early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.     

Preparatory changes and the development of the conjugation junction in a ciliate,Dileptus

Summary The present observations concern changes in the cortical structure of the ciliateDileptus prior to and during conjugation. The results can be summarized as follows: (1) Mating cells join each other in heteropolar configuration, i.e., the distal part of the oral apparatus of one partner faces the proximal part of the oral apparatus of another partner. (2) The structural changes prior to conjugation occur in the area of the oral apparatus. (3) The bonding area is situated within the oral apparatus, while the oral ciliature that encircles the oral apparatus remains outside the bonding area. (4) The fusion area is formed within the bonding area as a newly formed zone, without cortical organelles typically associated with the cell membrane. Two aspects of the formation of cell-to-cell union inDileptus are discussed: firstly, the heteropolar configuration of mating cells and its possible bearing upon an ability to form double cells when partners separation is inhibited. Secondly, the bonding area ofDileptus is compared to bonding areas of peniculine and hymenostome ciliates. A striking likeness in structural changes was found within the bonding areas of all three ciliates, in spite of the fact that the changes in question are localized within the oral apparatus ofDileptus, while inParamecium andTetrahymena the changed territory is situated between the oral apparatus and the anterior pole of the cell.     

The Exchange of RNA and Protein During Conjugation in Tetrahymena*

SYNOPSIS. Exchange of cytoplasm in Tetrahymena pyriformis, syngen 1, has been demonstrated by growing cells of 1 mating type in medium supplemented with H³-uridine or H³-histidine, washing, mixing with cells of an unlabeled, starved mating type, sampling conjugants at different times, and preparing autoradiographs. It was found that cytoplasmic interchange begins soon after the mates unite, and has become extensive before the end of the 1st prezygotic prophase (micronuclear crescent stage). When the RNA in one mating type had been labeled with H³-uridine, the activity was distributed almost evenly between the mates by late stages of conjugation. These results are consistent with electron micrographs of this syngen showing small pores in the attachment region of the mates, and many free ribosomes in the cytoplasm (8,11). By contrast, when protein in one mating type had been labeled with H³-histidine, these cells at late conjugation remained about twice as active as their originally unlabeled mates, presumably because of the physical characteristics of some structures which incorporated the amino acid (for example, cilia and membranes of the cell surface; cytoplasmic bodies, such as mitochondria, larger than the pores). That the radioactivity in the originally unlabeled cells came from their mates and not from the environment is indicated by the continued presence of inactive non-conjugants after 1 and 2 days in the mating type mixtures. Other cells which did acquire small amounts of active cytoplasm probably had engaged in abortive conjugation, separating from labeled mates before forming and exchanging pronuclei.     

Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

Changes in Oral Apparatus Structure Accompanying Vacuolar Formation in the Macrostomal Form of Tetrahymena vorax. A Model for the Formation of Food Vacuoles in Tetrahymena1

The large cytopharyngeal pouch of the macrostomal form of Tetrahymena vorax, following the addition of calcium, can form a sealed, empty vacuole. The open cytostomal region of this cell, which averages about 16 μ in diameter, is closed by an upward (ventral) movement of the right and posterior ribbed walls, both of which project into the cytostomal cavity. At the same time, the anterior and left walls of the cytostome-cytopharyngeal complex move to the right, forming a diagonally (right to left) placed furrow in the floor of the buccal cavity as these walls meet. As a result of the movement, the edges of the single membrane-bounded cytopharyngeal pouch are brought together and fuse, producing the closed vacuole. Elements of the cytoskeleton appear to participate in the closure process. Three major groups of ribbed wall microtubules support the open cytostome. The anterior ribbed wall microtubules pass laterally along the anterior (dorsal) portion of the cytopharyngeal pouch to the left where they end in the specialized cytoplasm. Middle oral rib microtubules terminate at the right and posterior margin of the cytopharynx while microtubules from the most posterior region of the ribbed wall pass to the left terminating in the specialized cytoplasm. The fine filamentous reticulum, a striated reticulum that borders the right, posterior, and anterior margins of the cytostome-cytopharyngeal complex, is in an ideal position to participate in these movements. It is anchored anteriorly high up in the buccal cavity to the cross-connective between the third membranelle and the undulating membrane complex. It courses beneath the right and posterior ribbed walls and runs laterally along the anterior margin of the cytopharynx to the left side. Contraction or pulling of this reticulum would act to bring the microtubule-reinforced walls of the cytopharynx together permitting fusion of the cytopharyngeal pouch membranes to form a sealed vacuole.     

Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila

Conjugating Tetrahymena were irradiated by ultraviolet-B (UV-B) at various stages of conjugation. When the conjugants were exposed to the UV-B at late meiotic prophase (the stage from pachytene to diplotene), abortive conjugation was induced at high frequencies. After completing meiosis, a significant number of the conjugants showed marked anomalies, i.e., failure of nuclear selection after meiosis, and abortion of the subsequent conjugation process such as a postmeiotic division to form gametic nuclei, nuclear exchange, synkaryon formation, and postzygotic development. The conjugating pairs retained the parental macronucleus and separated earlier as compared with a control. The resultant exconjugants degenerated meiotic products and became amicronucleates. These observations strongly suggest the presence of a UV-sensitive molecule that is expressed specifically at the meiotic prophase and that directs the subsequent development after meiosis. Dev. Genet. 23:151–157, 1998. © 1998 Wiley-Liss, Inc.     

The Cytology of Sheep Rumen Ciliates. II. Ultrastructure of Eudiplodinium maggii1

The anterior adoral zone of syncilia (AZS) of Eudiplodinium maggii is mounted on an extrusible peristome within a vestibulum. The peristome contains cytopharyngeal components derived from the infraciliature. These components include a crescent-shaped palisade of nematodesmata, two types of sub-membrane cytopharyngeal ribbons, and an ensheathing fibrous layer enclosing a phagoplasmic zone containing the other components. A convoluted esophagus is continuous with and extends from the posterior of the cytopharynx adjacent to the macronucleus. A posterior cytoproct has specialized cytoplasm around it and associated myoneme-like elements. The skeletal plate is composed of finely granular platelets and lies under the cortex ventral to the macronucleus. The endoplasm is separated from the ectoplasm by a fibrous boundary layer. The cortex has an external glycocalyx, a membranous layer, epiplasm, and microtubular and microfilament layers. The AZS infraciliature is of the usual cntodiniomorph type, kinetosomes linked by a sub-kinetosomal rod and with associated bifurcated kinetodesma, postciliary and transverse microtubules-the latter extending into the cytopharynx—nematodesmata, and a fibrous reticulum. A possible vestigial, somatic infraciliature consisting of short, barren kinetosomes with associated basal and cortex-directed microtubules and a periodic incomplete fiber, is found subcortically throughout the cell.     

Ultrastructure of the vegetative organisation and initial stages of silica plate deposition in the soil testate amoebaCorythion dubium

Summary The siliceous body plates ofCorythion dubium are bound by a band of organic cement which is thickest at the lateral margins. The anterior vacuolar cytoplasm is separated by a pigment zone, which forms a dark band in the mid-body region, from the compact posterior region containing a typical vesicular nucleus surrounded by a region of dense endoplasmic reticulum. A pellicular basket of microtubules surrounds the posterior cytoplasm. The large Golgi complex lies between the nucleus and the fundus. Numerous coated and uncoated vesicles from the Golgi cisternae are seen in the peripheral cytoplasm alongside developing plates. These small siliceous plates are enclosed in silicon deposition vesicles lying in surface ruffles of the plasmalemma, often in association with a pair of microtubules. Observations are made on the formation of these vesicles and the early stages of silica deposition. A comparison is drawn between silica deposition inC. dubium and choanoflagellates where there is a similar association between silicon deposition vesicles and microtubules.     

Mating Types, Breeding System, Conjuàtion and Nuclear Phenomena in the Marine Ciliate Euplotes cristatus Kahl from the Gulf of Naples

SYNOPSIS. The marine ciliate Euplotes cristatus Kahl (Ciliophora, Hypotrichida), collected off Capri, Gulf of Naples, is described in detail. From populations, 6 different mating types, representing 1 variety or syngen, have been isolated. The breeding relations revealed a multiple mating type system characteristic of other members of the Hypotrichida that have been investigated. Presumably a 7th mating type was found which does not mate with any of the others. Although this may belong to another syngen, it could represent a mating type which has not yet reached sexual maturity or 1 which may be in a period of decline. Animals of different mating type do not mate immediately after being mixed but usually 3 or more hours later. An agglutination reaction involving many specimens is absent. Instead, 2 ciliates engage in a “pairing play” before joining firmly in conjugation. Well-fed or actively feeding and dividing ciliates do not mate; mating occurs only after the food becomes gradually depleted or when the food supply is sharply cut off. All mating types appear to be extremely stable. Neither selfing pairs (intraclonal conjugation) nor autogamy have been observed within any clonal culture during the several years under investigation. Cell-free filtrates from 1 mating type do not elicit mating or induce conjugation with specimens of a different mating type. The general pattern of nuclear events in conjugation and exconjugant reorganization is as follows: 1 preliminary division, 3 pregamic (prezygotic) divisions, fertilization, and generally 1 or occasionally 2 postzygotic divisions. The fate of micronuclear products may be determined by their size and location. Those which are larger and close to the cell membranes of the joined conjugants persist and/or divide. Those which are smaller are carried by cyclosis toward the center of each ciliate and degenerate. The degenerating macronucleus of each conjugant becomes segmented in a more or less uniform manner resulting in 4 subspherical masses. Two become localized in the anterior end of a conjugant and 2 in the posterior end. Those in the posterior end are always the first to degenerate completely and disappear. In nuclear reorganization of the exconjugant, fusion of the macronuclear anlage with parts of the old macronucleus does not occur.