E. Marlo Nelsen

Development of the ciliature of Tetrahymena thermophila: I. Temporal coordination with oral development

Abstract The number of basal bodies and cilia along pole-to-pole ciliary rows was enumerated in Tetrahymena thermophila cells sampled during the rapid-exponential phase of culture growth in three different media that supported generation times ranging from 2 to 4 hr. The time required for oral development was nearly constant in the three media, and thus most of the differences in generation time were accounted for by differences in the interval prior to the onset of oral development (stage 0), which ranged from 50% of the generation time in the “poorest” medium to 20% in the “richest.” There was very little increase in number of basal bodies and of cilia along ciliary rows during stage 0, irrespective of the duration of this stage. The bulk of the increase took place during oral development, following a time course suggestive of coordination wth oral development. The same temporal pattern of increase was found in several ciliary rows, although the proportion of basal bodies that were ciliated differed among rows. There is no simple relationship between the number of basal bodies along ciliary rows and cell length, surface area, or volume. However, a large and constant proportion of the total division-to-division cell growth took place during the interval prior to the onset of oral development, suggesting that an ensemble of developmental events, including oral development and an associated activation of the remainder of the cell surface, may be triggered by attainment of a threshold cell size.

Mutations affecting cell division in Tetrahymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes.

Abstract The surface patterns associated with arrest of cell division by temperature-sensitive mutations at five different loci are described. Mutations at the mo1 locus prevent both the subdivision of ciliary meridians that mark the fission zone and the subsequent furrowing. Mutations at mo8 and mo12 cause abnormal configurations in the fission zone and aborted furrowing. Mutations in mo3 and mo6 bring about fission arrest with associated elongation ( mo3 ) or twisting ( mo6 ), even though complete fission zones do develop. The defects in mo1, mo3 , and mo12 are expressed in the first division after shift to restrictive temperature, whereas expression of mo6 and of one allele of mo8 are delayed. Following preincubation in an amino acid-free medium at the restrictive temperature, mo8 causes arrest at the first division after readdition of nutrients, while mo6 blocks fission only after one or more divisions at the restrictive temperature. Double homozygotes were constructed containing the mo3 a mutation and mutations at each of the other loci. In addition, mo1 a was combined with mo8 a . In each of the double homozygotes, the characteristic phenotypes of both mutations were simultaneously expressed, and the penetrance of division blockage was very greatly enhanced. The results suggest that the functions of these five loci are not ordered in a single dependent sequence of steps, but rather that these loci probably mediate independent processes required for cytokinesis.

Monoclonal Antibodies Reveal Complex Structure in the Membrane Skeleton of Tetrahymena

ABSTRACT. Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena. Five were directed against T. pyrifomis and seven were directed against T. thermophila. Some cross‐reactivity between species was found. Each monoclonal antibody recognized one of the three major components of epiplasm, i.e. the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins. It was found, using these antibodies, that the epiplasmic proteins A, B, and C have overlapping but independent distributions within the cell.

Transformation in Tetrahymena thermophila. Development of an inducible phenotype.

Abstract Tetrahymena thermophila transforms from a pyriform-shaped trophic form to an elongate rapidly swimming, dispersal form under the appropriate conditions of starvation [Nelsen, E. M., and DeBault, L. E. (1978). J. Protozool. 25, 113–119] . The development and control of the dispersal phenotype are examined. After an initial starvation period, the cell replaces its oral structures. During oral replacement, a caudal cilium emerges at the posterior end of the cell. As oral replacement is completed, the cell becomes spindle shaped and the newly-formed oral membranelles are positioned beneath the surface of the cell with somatic ciliary rows exterior to them. The spindle-shaped cell then elongates to become the dispersal form. While the cell is developing the new oral structures, it is also drastically increasing its numbers of somatic basal bodies and cilia. The events in the transformation pathway may be arrested or reversed by feeding the cell, except that once oral replacement has begun, it is completed along with an associated streamlining of the cell. Refed cells revert to the normal vegetative phenotype, except that some shape changes persist for several hours, suggesting that they are compatible with, but independent of, growth. Blockage of protein synthesis with cycloheximide prevents all changes associated with transformation, including the shape changes and elongation of the caudal cilium. The relation between transformation and conjugation has also been examined. Less transformation takes place when mating is possible, but transformed cells may also mate.

Fenestrin and Conjugation in Tetrahymena thermophila

ABSTRACT Certain monoclonal antibodies interact with proteins of Tetrahymena thermophila found in the conjugation junction as well as around the gametic nuclei (pronuclei) of conjugating cells; they also react with the oral primordium and fission zone of vegetative cells and with the cytoproct and contractile vacuole pores of all cells. One of these (FXIX‐3A7) was investigated in detail. Immunogold labelling suggests that the material labelled by the 3A7 monoclonal antibody, which we call “fenestrin,” is located beneath the epiplasm (membrane skeleton). Immunoblots reveal that the major and perhaps sole antigen is a 64 kDa polypeptide, found in two isoelectric variants. Developmental studies implicate fenestrin in two processes involved in conjugation. The first is “tip transformation.” During preliminary starvation (“initiation”), labelling of fenestrin first appeared as a spot at the anterior end of starved mature cells, then after mixing of different mating types (“costimulation”) it extended posteriorly along the anterior suture. After pairing, this region spread to form a widened plate. The second process is pronuclear transfer. Fenestrations representing channels between the conjugating cells began to appear 0.5 to 1 h after the conjugants united, and eventually merged to form a small number of temporary large holes during exchange of the transfer pronuclei. A fenestrin envelope also enclosed both the transfer and resident pronuclei; a strand of fenestrin connected the two. Shortly after pronuclear transfer, both transfer and resident pronuclei were released from fenestrin caps and fused to produce a zygotic nucleus (synkaryon) not associated with fenestrin. Fenestrin thus appears to be intimately involved in the process of pronuclear exchange.

Development of the ciliature of Tetrahymena thermophila: II. Spatial subdivision prior to cytokinesis☆

The cell surface of Tetrahymena thermophila is made up of an anterior region in which virtually all basal bodies of ciliary rows are ciliated, and the remainder in which ciliated and unciliated basal bodies are fairly irregularly interspersed. This pattern persists through interfission development until the stage of appearance of the equatorial ring of gaps in the ciliary rows that marks the fission zone. The ciliation pattern then becomes subdivided, in large part through the rapid ciliation of contiguous basal bodies located posterior to the fission zone. We interpret this process as a wave of ciliation of preexisting basal bodies that propagates posteriorly from the site of the fission zone. The location, extent, and timing of the ciliation process are the same in inverted as in normally oriented ciliary rows, in spite of the fact that in inverted rows the visible fission zone gap is tardily formed and the local configuration of ciliature around this gap is abnormal. The putative ciliation wave thus does not depend directly upon the local manifestations of the fission zone. However, in a cell-division-arrest mutant, cdaA1, analyzed under conditions in which formation of fission-zone gaps is permanently prevented in some ciliary rows but not in all, it is found that the ciliation pattern becomes subdivided in those ciliary rows that express fission-zone gaps and fails to become subdivided in neighboring rows that fail to manifest gaps. We interpret this combination of findings to indicate that a signal localized at the cell equator initiates a set of polarized developmental events that simultaneously create and demarcate two cellular fields within what was previously one. We further suggest that the characteristic tandem cell division pattern of ciliates is fundamentally a process of segmentation, which might involve mechanisms of gradient subdivision analogous to those taking place during segmentation of insects and other multicellular organisms.

Intracellular pattern reversal in Tetrahymena thermophila: I. Evidence for reverse intercalation in unbalanced doublets

Homopolar doublets of Tetrahymena thermophila possess two sets of similar cell surface structures, the most prominent of which are the complex and asymmetrical oral apparatuses. These initially are located on opposite surfaces of the duplex cell, but tend to shift so that they are no longer directly opposite each other. The two sets of oral structures are then separated by one wider and one narrower arc of cell surface. When one arc becomes sufficiently narrow, a new third oral apparatus with partially reversed internal asymmetry frequently becomes interposed between the two preexisting oral apparatuses, always within the narrower arc. After this happens, the reliability of development of new oral structures, particularly of the interposed ones, is reduced. Contractile vacuole pores, typically present within both arcs of homopolar doublets, tend to disappear from the narrower arcs. This anomalous partial triplet condition appears to be a transient intermediate stage in the reversion of homopolar doublets to normal singlets. We interpret the interposition of a transient third oral system in doublets that are regulating toward the singlet state as being a consequence of reverse intercalation of new positional values subsequent to excessive crowding of the preexisting positional values. This interpretation is an adaptation of the shortest-distance intercalation rule of the polar coordinate model applied in an intracellular and morphallactic context.

The Effects of Supraoptimal Temperatures on Population Growth and Cortical Patterning in Tetrahymena pyriformis and Tetrahymena thermophila: A Comparison

Abstract In this investigation, we compare the multiplication rates and morphogenetic responses of the two most studied Tetrahymena species, T. pyriformis and T. thermophila, at supraoptimal temperatures. Although the upper temperature limits differ greatly in the two species, the pattern of growth responses to high temperature is for the most part similar, with some differences in detail. The transient recovery of cell division at the highest temperature that allows cell division, characteristic of T. pyriformis, is observed in a less distinct form in T. thermophila. Moreover, there is a remarkable difference in developmental response, with drastic abnormalities in patterning of oral structures during the transient recovery of cell division in T. pyriformis, and far more limited abnormalities under similar conditions in T. thermophila. The abnormalities result from spatial disorder in the alignment and orientation of basal body pairs within the early oral primordium, followed by failures in the realignment that normally occurs as oral structures (membranelles and undulating membrane) mature. Both the initial spatial disorder and the failures in realignment are far more severe in T. pyriformis than in T. thermophila.

An Evaluation of Hsp90 as a Mediator of Cortical Patterning in Tetrahymena

Abstract This study asks two questions: 1) whether Hsp90 is involved in the regulation of cortical patterning in Tetrahymena, and 2) if it is, whether specific defects in this regulation can be attributed to functional insufficiency of the Hsp90 molecule. To address question 1, we compared the effects of a specific inhibitor of Hsp90, geldanamycin, on population growth and on development of the oral apparatus in two Tetrahymena species, T. pyriformis and T. thermophila. We observed that geldanamycin inhibits population growth in both species at very low concentrations, and that it has far more severe effects on oral patterning in T. pyriformis than in T. thermophila. These effects are parallel to those of high temperature in the same two species, and provide a tentative affirmative answer to the first question. To address question 2, we ascertained the base sequence of the genes that encode the Hsp90 molecules which are induced at high temperatures in both Tetrahymena species, as well as corresponding sequences in Paramecium tetraurelia. Extensive comparative analyses of the deduced amino acid sequences of the Hsp90 molecules of the two Tetrahymena species indicate that on the basis of what we currently know about Hsp90 both proteins are equally likely to be functional. Phylogenetic analyses of Hsp90 amino acid sequences indicate that the two Tetrahymena Hsp90 molecules have undergone a similar number of amino acid substitutions from their most recent common ancestor, with none of these corresponding to any known functionally critical region of the molecule. Thus there is no evidence that the Hsp90 molecule of T. pyriformis is functionally impaired; the flaw in the control of cortical patterning is more likely to be caused by defects in mechanism(s) that mediate the response to Hsp90, as would be expected from the “Hsp90 capacitor” model of Rutherford and Lindquist.