Cell-to-cell contact by locally differentiated surfaces in conjugation of Blepharisma

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium. When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.     

Mirror-imaged doublets of Tetmemena pustulata: implications for the development of left-right asymmetry

Ciliated protozoa possess cellular axes reflected in the arrangement of their ciliature. Upon transverse fission, daughter cells develop an identical ciliary pattern, ensuring perpetuation of the cellular phenotype. Experimentally manipulated cells can be induced to form atypical phenotypes, capable of intraclonal propagation and regeneration after encystment. One such phenotype in the ciliate Tetmemena pustulata (formerly Stylonychia pustulata) is the mirror-imaged doublet. These cells possess two distinct sets of ciliature, juxtaposed on the surfaces in mirror image symmetry, with a common anterior-posterior axis. We have examined whether individual ciliary components of Tetmemena mirror-image doublets are mirror imaged. Ultrastructural analysis indicates that despite global mirror imaging of the ciliature, detailed organization of the membranelles is reversed in the mirror-image oral apparatus (OA), such that the ciliary effective stroke propels food away from the OA. Assembly of compound ciliary structures of both OAs starts out identically, but as the structures associated with the mirror-image OA continue to form, the new set of membranelles undergoes a 180° planar rotation on the ventral surface relative to the same structures in the typical OA. The overall symmetry of the OA thus appears to be separable from the more localized assembly of individual basal bodies. True mirror imagery of the membranelles would require new enantiomorphic forms of the individual ciliary components, particularly the basal bodies, which is never observed. These observations suggest a mechanistic hypothesis with implications for the development of left-right asymmetry not only in ciliates, but perhaps also in development of left-right asymmetry in general.     

Pattern Formation in Mirror-Image Doublets of the Ciliate Stylonychia pustulata

ABSTRACT. Mirror-image symmetry doublets of the ciliate Stylonychia pustulata were obtained from the progenies of dividing cells in which cell division was inhibited by heat-shocks. In two components consisting of the doublet, the left (cell's) component possessed ciliary organelles arranged in almost the same pattern as in normal singlets, while the right one had surface organelles located in a mirror-image symmetry of those of the left component. In cell division of the doublet, two sets of ciliary primordia that were arranged in a mirror-image symmetry developed synchronously in both components. In about 80% of oral primordia (OP) of the right components, the arrangement of the membranellar bands became abnormal. In some cases, OP of the right component were occasionally separated into two longitudinal halves, each consisting of normal membranelles and inverted membranelles. A set of primordia of the paroral membranelles and fronto-ventro-transverse cirri was rarely derived from the basal bodies of the right half with a band of normal membranelles. As a result, a third component with the ciliary organelles normally arranged emerged on the right side of the original right component. The differentiation of membranelles and segmentation of the primordial streaks into cim proceeded from anterior to posterior. A cytoplasmic bulge with multiple right marginal cirral rows was frequently formed at the right margin of the doublet. The behavior in the separation of third and fourth streaks from a primordial streak of dorsal cirri was not mirror-image symmetrical in each component.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: starved versus nonstarved.

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Selective mirror-image reversal of ciliary patterns inTetrahymena thermophila homozygous for ajanus mutation

Summary The development of the oral apparatus (OA) and of neighboring ciliary structures ofTetrahymena thermophila was analyzed in cells homozygous for ajanus (jan A) mutation plus a recessiveenhancer of janA (eja). Such cells frequently possess two OAs located on opposite sides of the cell, a primary (1°) OA previously reported to be normal, and a secondary (2°) OA previously reported to express a mirror-reversal of right-left asymmetry. This study confirms the reality of a reversal in the gross orientation of membranelles in most developing 2° OAs. It also shows that there is a reversal of asymmetry in the pattern of resorption of basal bodies of ciliary rows adjacent to the 2° OA, and in the arrangement of basal-body couplets making up the portion of the apical crown of the cell situated close to the 2° OA. However, the locations at which membranelles of the 2° OA become modified during late phases of oral development remain normal, so that membranelles of 2° OAs are superimposable on those of 1° OAs. In addition, the membranelles of 2° OAs frequently undergo a rotation during the final phases of oral development, so that even their spatial orientation becomes normal. This mixture of reversed and normal features can be accounted for by postulating a superimposition of a reversed largescale asymmetry on a normal local asymmetry of ciliary units. This postulate predicts that no single mutation can bring about a complete mirror-image reversal of ciliary patterns.1° OAs appear normal by light microscopy. However, detailed analysis of SEM, preparations of isolated 1° OAs indicate subtle abnormalities of basal body arrangement in some of these OAs.     

Conjugation in the Ciliate Euplotes octocarinatus: Comparison of Ciliary and Cell Body-Associated Glycoconjugates of Non-mating-Competent, Mating-Competent, and Conjugating Cells

Pair formation in the hypotrichous ciliate Euplotes octocarinatus is a poorly understood phenomenon. In order to obtain information about the molecules involved in this process, we compared ciliary and cell body-associated glycoconjugates of non-mating-competent, mating-competent, and conjugating cells. Detection of glycoconjugates was carried out on Western blots by immunostaining of oxidized, digoxigenin-labeled carbohydrate moieties. Using this method, in both of two complementary mating types a 130-kDa glycoprotein was identified, which appeared on cilia during acquisition of mating competence and was reduced during cell pairing. This suggests an active role of this glycoprotein in ciliary adhesion during pair formation, Additionally, in both of the two mating types a cell body-associated 135-kDa glycoprotein was detected, which is present in non-mating-competent cells as well as in mating-competent cells, but is strongly reduced in conjugating cells. In contrast to the ciliary 130-kDa glycoprotein, the cell body-associated 135-kDa glycoprotein is not surface-exposed [8]. We therefore propose that the cell body-associated glycoprotein is either involved in the preparation for cell fusion or meiosis or that it serves as a cytoplasmic pool for the ciliary 130-kDa glycoprotein.     

Ultrastructure of the phallic glands of the firebrat,Thermobia domestica (packard) (Thysanura : Lepismatidae)

The exocrine glands located in the penis of Thermobia domestica (Thysanura : Lepismatidae) are composed of about 100 distinct units, each containing several cell types: one large secretory cell with an apical reservoir; 2 groups of envelope cells, an inner group of 2 superimposed cells, and an outer group of 4 cells arranged in a ring, and also 2 basal cells, called ciliary cells owing to their elongated processes, which look like the dendrite of a sensory cell. Each functional unit includes cuticular differentiations: a tubular bristle, fixed on a small tubercle; and a long “internal” ductule communicating basally with the reservoir of the glandular cell and opening distally at the tip of the bristle. A study of the modifications affecting the phallic glands during moulting, shows that the inner envelope cells deposit the cuticle that forms the ductule, the outer envelope cells elaborate the cuticule of the tubercle, while a temporary distal projection of only one of these cells ensures the formation of the bristle. In addition, a lengthening of the outer dendritic segment of the 2 ciliary cells takes place before ductule formation, but this segment partially degenerates after ecdysis. These findings are compared with data already obtained on the morphogenesis of other insect integumental glands. In T. domestica, the secretion of the phallic glands is presumed to be used, during the mating sequences, for spinning fine threads before spermatophore deposition.     

Scanning Electron Microscopy of Stomatogenesis Accompanying Conjugation in Euplotes aediculatus*

SYNOPSIS. The succession of morphologic changes in the feeding apparatus (peristome) accompanying conjugation and postconjugant development in the hypotrich Euplotes aediculatus has been examined by scanning electron microscopy (SEM). The details of stomatogenesis inferred from earlier light-microscopic studies of silver-stained preparations have been confirmed and extended. The elaborate peristome is the dominant surface feature of vegetative Euplotes. In conjugation, the ciliates are joined in their peristomial regions; as the conjugants separate, the old feeding apparatus is seen to be disrupted and partially resorbed. In its place is the crescent-shaped primordium of a new peristome, which develops as part of a general cortical reorganization. This primordium expands anteriorly, unfurling a new crown of ciliary membranelles that soon replaces the remaining preconjugant membranellar band. The resulting “exconjugant peristome'’is characterized by a greatly reduced number of adoral membranelles and the absence of paroral membranelles, buccal cavity, and cytostome. Exconjugants thus cannot feed for 2–3 days, until the missing peristomial components are replaced. This occurs by means of a 2nd cortical reorganization, during which new membranelles, developing from another peristomial rudiment, are added directly to the abbreviated exconjugant set. A new buccal cavity is concurrently sculpted as the primordial depression enlarges, and the cells can resume feeding sometime during the 4th day after separation. The implications of this mode of stomatogenesis and the nonfeeding condition are discussed, as are the advantages of SEM for studies of ciliate morphogenesis.     

Polypeptides during early conjugation in Tetrahymena thermophila

As Tetrahymena thermophila cells differentiate from their vegetative life cycle to sexual reproduction, their polypeptide pattern undergoes a series of changes. These changes have been traced in extracellular, cellular, and subcellular compartments. The first alteration is induced by the nutritional shift-down and results in stimulation of at least one ciliary polypeptide and affects a series of polypeptides from other compartments. The second alteration is induced by mixing starved cells of complementary mating types and this stimulates the synthesis of nine ciliary polypeptides before pairs have formed and eight afterwards. At least five of these early and one of the late conjugation-related ciliary polypeptides are removed by low concentrations of EDTA, indicating that they are located on the external side of the plasma membrane. No differences were observed between polypeptides excreted during starvation and after mixing of complementary mating types. At Tris concentrations restrictive for conjugation, cilia lack the conjugation-related polypeptides. Some of these are instead found among the excreted polypeptides. Using O'Farrell gels and silver staining on isogenic cells of all possible mating types, we have been unable to correlate changes in polypeptide patterns to specific mating types.     

ASPECTS OF CILIARY FINE STRUCTURE IN EUPLOTES PATELLA

1. The functional unity of cirri and membranelles can result structurally only from extensions of the ciliary membrane. 2. The pellicle is composed of an outer pellicular membrane and an inner cytoplasmic membrane. 3. The ciliary rootlets are composed of numerous filaments 120 A in diameter with central areas of low density. They have no periodic structure. 4. The ciliary membrane is a double-layered structure continuous with the pellicular membrane. The cilia show the typical arrangement of nine double, peripheral and two single, central fibrils. All fibrils pass into the basal region, the peripheral ones joining with the rootlet filaments, while the central fibrils from the extreme proximal position of the basal region turn back toward the pellicle and appear to unite just beneath the cytoplasmic membrane. 5. The cilia (300 mµ diameter) taper at their tips to a diameter at least as small as 50 mµ. At a diameter of about 150 mµ, the fibrils begin to show a reduction in number. 6. The central ciliary fibrils may determine the possible directions of ciliary beat. These fibrils show an intrafibrillar structure in their basal portion, which involves regularly spaced 40 A granules. 7. These observations on Euplotes, together with the other evidence cited, are consistent with the hypothesis that ciliary motion is produced by the contraction of the peripheral fibrils, while the central fibrils perhaps determine the plane in which the cilia can bend.     

The filaments supporting ciliary primordia and fission furrow in the hypotrich ciliateParaurostyla weissei, revealed by the monoclonal antibody 12G9: Studies on wild-type and ciliary hypertrophy mutant

Summary The unique monoclonal antibody FXXXIX 12G9 obtained againstTetrahymena cortices was used to label cytoskeletal structures related to basal body proliferation inParaurostyla weissei. The antibody binds to an amorphous material interconnecting basal bodies in compound ciliary structures: dorsal units, cirri and membranelles in interfission cells, and filamentous structures supporting the primordia of ciliary structures and fission line in dividing cells. The antibody visualized meridional filaments preceding proliferation of new basal bodies in the oral primordium and structures accompanying all developing ciliary primordia. It congregated in differentiating new procirri and membranelles, whereas another population of transient meridional structures accompanied the final distribution of new structures. A meridional filament connecting transverse cirri with the oral apparatus, marking the future stomatogenic meridian, persisted in both division products until completion of cell elongation. The fission line was found to originate from an anterior extension of the pre-oral filament toward the parental oral structures. It then encircled the cell's midbody demarcating the boundary between daughter cells; two additional circumferential structures bordering the anterior and posterior ends of differentiating division products participate in formation of the new poles. They disappear after separation of daughter cells and completion of resorption of parental ciliature. In the enhanced multi-left-marginal mutant expressing gross hyperduplication of basal bodies, the location of the 12G9 antigen corresponded to that in wild-type cells. The sequence of formation of meridional filaments in the mutant was found to be altered. The filaments in the left lateral domain preceded the formation of the preoral filament, yet the temporal pattern of basal body assembly was not modified. The fission line, as in wild-type cells, originated in connection with the oral primordium. We conclude that the nucleation of the filamentous structures bearing the 12G9 antigen and the basal body assembly occur by independent mechanisms reading the same cell cycle signals. We suggest that the 12G9-antigen-bearing protein might be similar to septins: involved in signaling the position of the oral primordium and the fission line and functioning in establishing and maintaining the asymmetric cortical domain characteristics.Abbrevations AZM zone of adorai membranelles - bb basal bodies - CC caudal cirri - FC frontal cirri - Fmf frontal meridional filament - FTV the primordia of fronto-ventro-transverse cirri - LD, RD dorsal rows of bristle units - LM, RM left or right marginal cirral row - OA oral apparatus - OP primordium of the adoral membranelles - pLM, pRM primordium of the left or right marginal cirri - pLD, pRD primordia of the left or right dorsal bristle rows - pUM primordium of the undulating membranes - TC transverse cirri - UM undulating membranes - VC ventral cirral rows     

A Single-Gene-Dependent Abnormality of Adoral Membranelles in TETRAHYMENA PYRIFORMIS, Species 1

Strain D of species (syngen) 1, Tetrahymena pyriformis, differs from other inbred strains in its manifestation of certain abnormal patterns of adoral membranelles. Instead of the usual three membranelles some cells have a greater number, most frequently 4 or 5, but occasionally up to 7. The extra membranelles, or even all membranelles of any given set, are shorter than M-1 and M-2 of the normal pattern. In other cases, the only alteration observed is a change in the relative lengths of the three membranelles. The frequency of abnormal cells varies from about 5% to 15% during exponential growth to over 50% after prolonged stationary culture. The genetic basis for the abnormality is shown to be due to a single recessive gene which segregates normally in various crosses and which manifests vegetative assortment as do most allelic variants in species 1.     

棘尾虫对折右纵断片的再生和纤毛模式形成的研究

应用切割嫁接方法获得的棘尾虫完全对折和部分对拆右纵断片都经历了一面调整极性、一面再生和形态发生的过程。推测这两种断片在纤毛模式形成中发生的诸多现象可能与所在断片再生时的极性调整变化状态有关。     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

Oral assembly in left-handed Tetrahymena thermophila

We have investigated oral development in a non-genetically derived left-handed (LH) form of Tetrahymena thermophila, in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells' anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180 degrees rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama. We present a model for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

Development of the Ciliary Pattern of the Oral Apparatus of Tetrahymena thermophila1

ABSTRACT. The sequence of formation and ciliation of basal bodies and the subsequent organization of compound ciliary structures of the oral apparatus of Tetrahymena thermophila was reanalyzed with the aid of scanning electron microscopy of cells in which the epiplasmic layer was exposed, as well as by light microscopy of protargol-impregnated specimens. This combination of methods allowed the delineation of numerous steps in the patterning of the oral ciliature, some of which have received little or no previous attention. Highlights include: the initial formation of “strings” of nonciliated new basal bodies in juxtaposition to relatively few basal bodies of the stomatogenic kinety; generation of basal body pairs, roughly oriented along the anteroposterior axis of the cell, that later align side-by-side to assemble promembranelles; condensation and reorientation of promembranelles simultaneous with addition of a third row of basal bodies anterior to the original two rows; production of a very short fourth row of basal bodies at the anterior right end of each developing membranelle; generation of the outer basal body row of the undulating membrane (UM) after alignment of the inner row, with transient ciliation of the inner row preceding permanent ciliation of the outer row; limited basal body resorption at the ends of membranelles; and sculpturing of the right ends of membranelles by a movement of basal bodies associated with formation of the ribbed wall adjacent to the UM. In the old anterior oral apparatus a repetition of the processes of generation of a new outer UM row and sculpturing of right ends of membranelles takes place in synchrony with the corresponding events in the oral primordium, following prior shedding of the old outer UM row and loss of the sculptured pattern in association with temporary regression of the ribbed wall micro-tubules. Oral development is complex, with different processes involved in the assembly of the membranelles and the UM, and with a sequence of distinct events involved in the generation of each of these structures. Speaking comparatively, membranelle development follows the same pathway in many, perhaps all, ciliates in which these structures or their homologues develop from a common stomatogenic field.