Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.     

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible “attack me” signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.Key words: Tetrahymena, conjugation, nuclear apoptosis, monodansylcadaverine, macroautophagy, phagocytosis marker, glycoconjugates, phosphatidylserine     

Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.     

The intranuclear helices of Euplotes: Their substructure,localization, and apparent migration to the cytoplasm

Study of the fine structure of the macronucleus in Euplotes eurystomus, a ciliate protozoon, during various stages of the cell division cycle has yielded new information about intranuclear helices. They are frequently observed at the periphery of chromatin bodies or next to the nuclear envelope, and they appear to be a constituent of nucleoli. The fibril that forms a helix is about 11–15 nm thick, and torus profiles of helices cut in cross section are about 35 nm in diameter. In substructure the helix is composed of a thin strand 3–5 nm thick which is coiled to form the 11–15 nm fibril; so the helix is a super-coiled structure. The intranuclear helices are present in the macronucleus throughout the cell cycle. They do not show obvious changes of relative abundance nor changes of relative localization in the nucleus, with one exception: they were never observed in the diffuse zone of replication bands. Evidence is presented indicating that nuclear helices migrate to the cytoplasm through nuclear pores. Although the chemical composition of the Euplotes intranuclear helices is unknown, information in the literature on similar helices in Amoeba indicates that they contain RNA and not DNA. The observations on Euplotes helices are consistent with a concept of “packaged” RNA for transport to the cytoplasm.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

Resting Cysts of the Pigmented Ciliate Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea)

The morphology of Blepharisma sinuosum resting cysts and the dynamics of pigmentation at different stages of encystment are presented for the first time. Cyst morphometrics are similar to other Blepharisma species, with three‐wall layers, bacteria surrounding the ectocyst, a conical plug, and wrinkly surface toward the plug in mature stages. The vegetative moniliform macronucleus changes to a horseshoe shape, and at early stages, the cystic cytoplasm is homogeneously pigmented, comprising a contractile vacuole; later, pigments polarize toward the plug, decorate the cortical layer, and become brownish. This work reinforces the potential role of pigment dynamics on cyst biology.     

Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

冠突伪尾柱虫（Pseudourostyla cristata）实验再生研究

冠突伪尾柱虫(Pseudvurostyla cristata) 含约70枚大核。我们用显微手术横切G1期细胞,得前后两块相等断片;分别培养。60小时后,断片再生完成。在再生过程中,随细胞体积增大,大核数目也增加。大核的数目和细胞体积存在着一定的均衡关系。在细胞无性分裂过程中,许多大核改组后,融合成一个融合大核。这个融合大核具两个仔虫的大核数目和DNA量。我们用显微手术得到含融合大核的后断片。在后断片再生后恢复的虫体内,我们发现本应分配到两个仔虫中去的大核数目,被限制在一个虫体的大核数目上。这说明了细胞质可以影响和调节大核的数目。并还证明了这种虫体大核DNA量较正常虫的大核DNA量约多一倍。其中大部分虫体分裂时,大核不经改组就开始融合和分裂;从而使DNA量回复正常。同讨还发现小部分虫体通过排出大核多余核物质方式来调节大核DNA量。这些现象说明了细胞核质之间存在着一种调节相对平衡和相互协调的机制。     

Ultrastructure of erythrocytes and leucocytes of Priapulus caudatus (de Lamarck) (Priapulida)

The marine priapulid Priapulus caudatus has a voluminous body cavity filled with a blood-like fluid containing erythrocytes and leucocytes (amoebocytes). The hematocrit of animals weighing 0.5–14 gm was 2–10%. The erythrocytes contain a hemerythrin blood pigment. The structure of the coelomocytes was studied by light and electron microscopy. The erythrocytes are nucleated and contain marginal bands, vacuoles and occasionally crystals. The cytoplasm has few organelles. The leucocytes are amoeboid motile cells, the cytoplasm of which contains numerous organelles. The most conspicuous of these are oval particles, probably representing developmental stages of lysosomes. Most of these organelles contain tubules stretching from one pole to another. In the hind part of the animal, certain tissues, primarily the posterior warts contain large numbers of coelomocytes. The histological picture is complicated, showing some resemblance to the lymphoepithelial tissues of vertebrates.     

FROM PROTOPLASM TO SWARMER: REGENERATION OF PROTOPLASTS FROM DISINTEGRATED CELLS OF THE MULTICELLULAR MARINE GREEN ALGA MICRODICTYON UMBILICATUM (CHLOROPHYTA)1

Protoplast regeneration from extruded cytoplasm of the multicellular marine green alga Microdictyon umbilicatum (Velley) Zanardini (Cladophorales, Anadyomenaceae) was investigated. The early process of protoplast formation is comprised of two steps: agglutination of cell organelles into protoplasmic masses followed by generation of a temporary enclosing envelope around them. Agglutination of cell organelles was mediated by a lectin–carbohydrate complementary system. Three sugars, D‐galactosamine, D‐glucosamine, and α‐D‐mannose, inhibited the agglutination process, and three complementary lectins for the above sugars, peanut agglutinin, Ricinus communis agglutinin, and concanavalin A, bound to the surfaces of chloroplasts. Agglutination assay using human erythrocytes showed the presence of lectins specific for the above sugars in the algal vacuolar sap. A fluorescent probe 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐a, 3,5‐hexatriene revealed that the envelope initially surrounding protoplasts was not a lipid‐based cell membrane. However, this developed several hours later. Simultaneous fluorescein diacetate and propidium iodide staining showed that the primary envelope had some characteristics of cell membranes, such as semipermeability and selective transport of materials. Also, fluorescein diacetate staining showed esterase activity in the protoplast and relocation of cell organelles and compartmentalization of cytoplasm during the process of regeneration. Both pH 7–9 and salinity 400–500 mM were found to be essentially important for the development of the protoplast envelope. When the basic regeneration process was accomplished, two alternative pathways of development were seen; about 70% of one‐celled protoplasts transformed into reproductive cells within 2 weeks after wounding, whereas others began cell division and grew into typical Microdictyon thalli. Quadriflagellate swarmers were liberated from the reproductive cells, and they germinated into mature individuals. It is therefore suggested that this species may use the wound response as a method of propagation and dispersal.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Nuclear Activity During Regeneration in Blepharisma intermedium Bhandary*

SYNOPSIS. Transection and regeneration of Blepharisma intermedium initiate complex macronuclear activity and micronuclear division. Reversible condensation of the macronucleus is achieved by contraction which involves primary and secondary coiling. Primary coiling, evident in vegetative cells, is enhanced by pronounced constriction of the macronuclear extremities during regeneration. Secondary coiling is restricted to the period of contraction and is initiated at the ends of the mauonucleus. Condensation terminates the coiling processes and gives rise to an amorphous structure. Re-elongation of the macronucleus is not followed immediately by coiling. Micronuclear division begins early in regeneration, but the peak of visible activity is not reached until the 3rd through 5th hours.     

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila,as detected by in situ hybridization

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with ³H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei had disappeared. The hybridization in the anlage then decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.     

Ultrastructural observations on gastrodermal regeneration in the planarian Dugesia japonica (Turbellaria)

The earliest detectable change during regeneration of the gastrodermis in Dugesia japonica was an aggregation of regenerative cells underneath the gastrodermis remaining at the wound margin. The gastrodermal cells in experimental regenerates retained some of their original characters and presented no indication of cell dedifferentiation. The regenerative cells came into contact with the basal surface of gastrodermal cells, forming stratified cell layers. Differentiation of these cells into gastrodermal cells was initiated by the development of synthetic organelles within their cytoplasm. These differentiating cells gave rise to two different types of gastrodermal cells, namely phagocytic cells and sphere cells. In later stages, there was an apparent movement of differentiated gastrodermal cells towards the parenchyma.     

Characterization of the Life Cycle and Heteromeric Nature of the Macronucleus of the Ciliate Chilodonella uncinata Using Fluorescence Microscopy

Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA‐rich and DNA‐poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4′,6‐diamidino‐2‐phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life‐cycle stages. We photo‐documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z‐stacks and three‐dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.     

The Fine Structure of the Phoront of the Apostomatous Ciliate,Hyalophysa chattoni*

The phoront of the apostomatous ciliate, Hyalophysa chattoni, is an encysted stage that is carried on the exoskeleton of its crustacean host until the ecdysis of the host. At molting the phoront rapidly metamorphoses to the feeding stage, excysts, and immediately begins to feed on exuvial fluid trapped in the cast-off exoskeleton. The fine structure of the resting phoront resembles that of the preceding migratory stage, the tomite. A prominent ventral tuft of cilia, the ogival field, has vanished, and the trichocysts that paralleled the kinetics have disappeared. The dense inclusion bodies that were concentrated around the mouth and falciform fields have dispersed and greatly decreased in number. The cytoplasm and its membranous organelles do not appear visibly condensed or altered from the preceding stage in the life cycle. The phoront is merely quiescent instead of dormant. Unlike the few ciliate cysts previously examined by electron microscopy, the phoront's cyst is not divisible into separable layers. It resembles the loricae of certain suctoria in being formed principally of a fibrous substance, the outer surface of which has a paracrystalline pattern. The peduncle attaching the cyst to the crab's gill is a continuation of the cyst wall although its structure is somewhat modified. The most conspicuous innovation in the phoront's fine structure is the massive tracts of microtubules that run longitudinally through the macronucleus. The microtubules are in intimate contact with Feulgen-positive chromatin masses which are crowded toward the periphery of the macronucleus.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post-conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non-irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.