The giant of Oxytricha bifaria: A peculiar cell differentiation triggered and controlled by cell to cell contacts

European Journal of

Europ.]. Protistol. 27, 127-133 (1991) June 21, 1991

PROTISTOLOGY

The Giant of Oxytricha bifaria: A Peculiar Cell Differentiation Triggered and Controlled by Cell to Cell Contacts' Nicola Ricci, Giuseppina Grandini, Annalisa Bravi and Rosalba Banchetti Dipartimento di Scienze dell'Ambiente Italy

e del Territorio, Gruppo di Protistologia, Pisa,

SUMMARY The heteromorphic life cycle of the freshwater hypotrich Oxytricha bifaria comprehends at least 3 phases: conjugating pairs, resting cysts and carnivorous giants. The gigantic forms represent unique adaptive devices which enable the species to survive in a certain environment when the normal bacterial food is exhausted. In this report several results are described which deal with the nature of the mechanisms underlying giant formation: (a) only cell to cell contacts (not species-specific)occurring among living specimens trigger this differentiation; (b) the higher the number of cellsper ml, the shorter the "induction period", namely the time lag between the onset of the inducing conditions and the formation of the first giant; (c) it is now possible to separate and to distinguish experimentally, within the "induction period", two successive steps, namely "activation" and "predation"; (d) also the already-differentiated-giants (the so-called steadystate-giants) need cell to cell contacts to maintain their cell differentiation. The "Labile Memory Counter" hypothesis is proposed and discussed.

Introduction Many protozoa, eukaryotic unicellular organisms, can dramatically and consistently change their morphology and physiology (i.e., they can differentiate) to match the challenges periodically met either in the most severe bottlenecks of their life cycles (as sexual phenomena, for instance) or in their environment (severe physico-chemical conditions). How is this differentiation controlled? In metazoan developmental processes at least two strategies have been adopted to guide the differentiative story of a cell or of a group of cells as suggested by Grobstein as early as 1956: (a) long-range interactions mediated by soluble inductors such as those involved in metamorphosis of Amphibians [31] and of Insects [6,8], (b) short-range interactions (cellcontacts: d. Jacobson [13] and Muthukkaruppan [20] for the mouse lens development, Grobstein 1

This work was supported by funds from CNR and MURST.

© 1991 by Gustav Fischer Verlag, Stuttgart

[10] for the development of mouse metanephros, Slavkin and Bringas [33] for the odontogenesis, Cutler and Chaudhry [3] for the development of the rat submandibular gland, Lehtonen et al. [15] for the formation of kidney tubules). Quite similar is the case of protozoa, whose differentiative biology may rely on soluble factors (d. Miyake [17] for Blepharisma japonicum; Kochert [14] for Volvox carteri; O'Day and Lewis [21] for Dictyostelium discoideum), on direct cell contacts (d. Goodenough [7] for Chlamydomonas; Hiwatashi [12] for Paramecium), or even on both systems [22]. Oxytricha bifaria is the hypotrich ciliate investigated in our Lab from the cell differentiation point of view over the last two decades, because its life cyclepresents at least three states characterized by clearcut morphophysiological changes: pairs, cysts, giants. The conjugating pair, in which the sexual processes occur, is formed only when several successive steps are properly accomplished [22]. A mild starvation represents the environmental trigger of the whole process: the 0932-4739/91/0027-0127$3.50/0

128 . N. Ricci, G. Grandini, A. Bravi and R. Banchetti

following first recognitions among potential partners are mediated by mating-type specific soluble factors, gamones (long-range strategy) [4], while later stages of these preconjugant cell interactions require topographically and chemically proper heterologous cell-cell contacts, to trigger both the cell membrane fusion and the induction of meiosis in the partners (short-range strategy) [25, 26]. As to the occurrence of the encystment a still unknown combination of environmental parameters (temperature, oxygen dissolved, pH...) seems to work as the actual factor setting off the process [9]. The gigantic, carnivorous forms are easily recognizable, even at very low magnification [27]. They are produced by O. bifaria to shift for a certain diet (bacteria) to a new one (other ciliates), after the bacterial food is exhausted: in this way they can exploit a certain favorable micropatch for a longer period of time than that of the normal oxytrichas [28]. The dramatic morphological [32] and nuclear changes [30] have been described and found to be triggered by cell-cell contacts, which are not species-specific [28]. To obtain a more complete picture of the differentiation potentialities of O. bifaria, several experiments were carried out on the nature of the cell-cell contacts responsible for the differentiation itself: do they represent a sufficient condition for it to occur, or just a necessary one? Do they play any role in the maintenance of the already differentiated forms? May any unifying working hypothesis be proposed to account for the different aspects of the process we know?

Experiment 3: Mechanic Perturbation of the Inducing Conditions Experimental populations of S 9 strain (800 cells/ml, 10 ml in 25 ml beakers) were treated in a magnetic stirrer, which whirled the whole volume (= no still cell anywhere) without damaging the cells.Similar, not stirred populations were used as controls. When the first giant formed in the controls, the stirrer was switched off and the induction period measured. In a second round of experiments the same protocol was used for different populations at different cell densities and the different induction periods measured. .

Experiment 4: Biological Simulation of the Inducing Conditions B. [aponicum, R 103 strain, was cultured according to Miyake et al. [19] and used to activate underthreshold populations of O. bifaria strain S 9 (9 depressions; 1 mlldepression); 9 depressions with 150 oxytrichas/ml were used as underthreshold controls. In a second type of experiment three populations were studied: (i) controls; (ii) 5000 blepharismas/ml with 50 oxytrichas/ml (20 ml); (iii) 50 oxytrichas/ml (20 ml); when in (i) the first giant formed, the (ii) oxytrichas were isolated, freed of B. japonicum, concentrated up to = 400 cells/ml and the induction period was measured; at the same time the population (iii) was also concentrated to 400 cells/ml and observed to measure its induction period. In a third type of experiment different densities of B. japonicum (5000,2500, 1200, 600, 300 blepharismas/ml) were incubated with 150 oxytrichas/ml: the induction period was measured in 4 depressions/treatment.

Experiment 5: Cell Contacts and Steady State Giants Material and Methods The strains S 6 and S 9 of O. bifaria Stokes (1889) cultured according to Ricci et al. [24] were used. These strains form giants only under crowding conditions: the threshold values of cell density (namely those below which no giants differentiate) are of 300 cells/ml for S 6 and 220 cells/ml for S 9. The cells were collected by a mild centrifugation (= 180 g) and then handled and observed according to the procedures already reported [27,28]. Throughout the experiments the temperature of 23 DC was maintained; only the experiment 2 (see below) was carried out at 19 DC. The following descriptions account for the new, more specific methods.

Experiment 1: Killed Oxytrichas as Potential Inducers of Giant-formation Underthreshold (150 cells/ml) populations of O. bifaria were exposed to two inducing agents: (a) frozen-thawed oxytrichas, (b) oxytrichas fixed by K2Cr207 (0.2 M, [18]). Either treatment was carried out at two cell densities of inducing killed cells (1500, 3000 cells/ml).

Severalpopulations of O. bifaria, strain S 9, were concentrated to obtain a large amount of giants after 36 h. Once the giants are formed, they feed on other ciliates, grow and undergo periodic binary fissions [27]: these giants are defined "steady state giants". SSG were transferred to different media and isolated one per depression: 9 in 5MB (SyntheticMedium for Blepharisma) [17],9 in lettuce medium, 9 in food (lettuce medium inoculated with Enterobacter aerogenes 24 h earlier), 9 in food containing O. bi[aria (150 cells/ml). The fate of the different groups was studied. In a second type of experiment SSG were treated (a) with concanavalin-A (Con-A) (Sigma,n. C-201O) and then washed free from this lectin and (b) with Con-A plus a-methyl-D-mannoside (Sigma, n. M-3752), as a control [28]. Both a- and b-SSG were then isolated one per depression in food containing 150 oxytrichas/ml and their fate was studied. In a third experiment SSGwere isolated in different depressions containing O. bifaria (pretreated with Con-A and then washed free from the lectin) at different cell densities: 4000, 2000, 1000 and 500 oxytrichas/ml. This treatment was also used with SSG isolated in the same series of experimental populations of O. bifaria pretreated with Con-A plus a-methyl-D-mannoside, as controls for the Con-A treatment.

Experiment 2: Cell Density and the Induction Period S 6 and S 9 strains were concentrated up to 5000 cells/ml and 4000 cells/ml, respectively: a series of progressive 1 : 1 dilutions was then made and the different populations were scored to measure the time lag elapsing between the onset of the overthreshold conditions and the appearance of the first giant: this parameter was called "induction period". These experiments were run at 19 DC (Fig. 1).

Results Experiment 1: Killed Oxytrichas as Potential Inducers of Giant Formation Dead oxytrichas (= 1500 cells/ml) were used to induce living underthreshold populations to produce giants. The

Cell Contacts in Giants of Oxytricha . 129 induct ion p e r iod 40

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Fig. 1. The effects of cell density on the induction period of the two strains S 6 (A-A) and S9 (0-0). 20

results showed that neither frozen-thawed nor mildly fixed oxytrichas can trigger this phenomenon, regardless of their cell density. Experiment 2: Cell Density and Induction Period Length The role of the cell density in determining the time lag preceding the differentiation of the very first giants (= induction period) was studied. The results of only one experiment, out of the 6 carried out, are shown in Fig. 1, the variations among them being statistically irrelevant: (a) the higher the number of cells/ml, the shorter the induction period; (b) the induction period cannot be shortened beyond a certain minimum value, whose absolute value is definitely strain-dependent: = 9 h for S 6 and 5 h for

S 9.

Experiment 3: Mechanic Perturbation of the Inducing Conditions Experimental overthreshold populations (800 cells/ml) were stirred for 8 h, i.e. for a time corresponding to the induction period of the control populations: within 3 h from the end of stirring, the first giants formed in these experimental populations (Fig. 2), thus showing that some kind of "activation" had occurred during the stirring conditions, which "prepared" the cells to prey on other

giants (%)

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12

14

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Fig. 3. The same experiment as shown in Fig. 2, run with higher cell densities. When the controls (..- ..) formed the first giant (induction period = 10 h), the stirrer was stopped (arrow) and the different populations observed for their induction periods: for 6000 and 5000 cells/ml (induction period = 40') the results overlapped perfectly (0-0) so that only one curve is given; for 3000 cells/ml (induction period = 60') the trend of the curve (e-e) was a little lower.

oxytrichas as soon as the mechanical disturbance finished. When the number of cells per ml of the stirred populations was strikingly increased (3000,5000 and 6000 cells/ml), the time required for the formation of the first giant decreased progressively from 1 h, to 40 min and again 40 min, respectively, for the 3 cell densities tested (Fig. 3). Experiment 4: Biological Simulation of the Inducing Conditions The results of these experiments are shown in Figs. 4, 5, 6: the use of B. japonicum as living experimental "stimulator" for O. bifaria's giant formation in underthreshold populations (150 cells/ml) demonstrated the following points: (A) B. japonicum can stimulate a certain, underthreshold population of O. bifaria so that it produces giants (Fig. 4);

60

(B) this treatment is completely physiological (no irregular cell ever observed), but not as effective as the already described experimental stirring, being incapable of reduc-

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Fig. 2. The activation of O. bifaria, obtained by magnetic stirring. The stirrer was switched off (arrow) when the first giant appeared in controls (..- ..: induction period = 8 h). The induction period of stirred populations (0-0) was about 3 h.

10

15

20

2 5 3 0 time (hr)

Fig. 4. Underthreshold populations of O. bifaria (150 cells/ml) (..- ..) may be stimulated to produce giants, by contacts with B. japonicum (600 cells/ml) (0-0).

130 . N. Ricci, G. Grandini, A. Bravi and R. Banchetti 100

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Fig. 7. While SSG isolated in populations of O. bifaria (150 cells/ml) keep dividing regulatly (controls : EB-EB), SSGisolated in acellular media dedifferentiate, returning to the normal morphophysiological state typical of the species within a certain time: in bacterized food (6-6: 15 h), in 5MB (0 - 0 :22 h) and in autoclaved lettuce medium (A-A: 24 h).

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Fig. 5. The normal kinetics of giant formation of a control population (400 oxytrichas/ml) (.A.-A) compared with the other two experimental ones; after 10 h (arrow), underthreshold popul ations of O. bifaria (50 cells/ml) were freed from the inducing B. japonicum (5000 cells/ml) and concentrated at the same cell density as the controls and their kinetics studied (0-0); at the same time, other underthreshold populations of O. bifaria (50 cells/ml) (which did not interact with Blepharisma) were concentrated at the same cell density and their kinetics observed as a control of this kind of experimental treatment (e-e): their induction period (= 8 h) was very similar to the controls (= 7 h) both being more than twice as much as th at of the experimental popul at ions (= 3 h).

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Fig. 6. As the cell density of B. japonicum increases from 300 to 5000 cells/ml, the inductive effect on underthreshold populations of O. bifaria (150 cells/ml) increases: beyond a certain value (= 2500-3000 cells/ml) the trend reverts.

ing the induction period to values shorter than three hours (Fig. 5 ); (C) the Blepharisma-Oxytricha cell-cell contacts, which

proved to elicit the induction of giants, demonstrated that for this process neither species-specific contacts nor preypredator contacts are necessar y to activate the oxytri chas; (D) the predatory behavior represents the second necessary step of the process of giant formation; (E) the inducing effect of Blepharisma cells depends upon their number per ml: beyond a certain upper limit (2500 cells/ml), however, this positive correlation occurring between Blepharisma cell density and Oxytricha differentiati on kinetics reverts (Fig. 6).

Experiment 5: Cell Contacts and Steady State giants Steady-state giants, isolated one per depression in different media (bacterized food , 5MB and autoclaved lettuce medium) divide and return to the normal morphophysiological condition of the species, by means of 3 binary fissions occurring in different time lags for the 3 experimental conditions, namely 16 h, 22 h, 24 h respec-

ded iffe ren tia ting c.II1

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Fig. 8. The experimental SSG treated with 3 ug/ml Con-A and 40 ug/ml a-methyl-D-mannoside for 2 h (0- 0 ) behave as the controls (EB-EB); the SSG treated with 3 ug/ml Con-A for 2 h in presence of oxytrichas as potential food (A-A) de-differentiate in much the same way as the giants in presence of oxytrichas pre-treated for 2 h with 3 ug/ml Con-A (6-6) .

tively. The controls, kept in underthreshold populations (150 cells/m!) of O. bifaria, keep dividing regularl y as giant s (Fig. 7). This result shows that (a) the active presence of cell-preys is necessary to maintain the differentiated state and that (b) also for the dedifferentiation proce ss a certain energetic input is required: the giants feeding on bacteria (Fig. 7, L'-,- L'-,) , indeed, de-differentiate faster than those in 5MB (Fig. 7, 0-0) and in lettuce medium (Fig. 7, . - .). In Fig. 8 the results of Con-A treatment of SSG are shown: (1) the lectin specifically interfere s with the process of steady state maintenance, being inhibited by a-methylD-mannoside; (2) both SSG pretreated with Con -A in presence of normal oxytrichas and normal SSG with oxytrichas pretreated with Con-A dedifferentiate. These results indicate that both the prey and the predator must contact each other physiologically (namely by means of

Cell Contacts in Giants of Oxytricha . 131 their normal cortical apparati), also to enable the steady state giants to maintain their differentiation. Discussion The general outcome of the round of experiments here reported on the role of cellular contacts in triggering the differentiation of giants in populations of o. bifaria are in quite good agreement with what is already reported (no detectable soluble factor) on the basis of the results by Ricci et al. [28]: both the induction of giants by magnetic stirring (Exp.3) and by B. japonicum cells (Exp.4), indeed, not only confirmed that the maximum number of giants formed in a certain population depends upon its cell density [27], but also demonstrated that the induction period also depends, at least up to a certain value, upon the cell density of that population. If we now consider that o. bifaria was shown to rely on highly specific cell-cell contacts to bring about its sexual processes (conjugation) [22, 25] both at the level of cell fusion and of meiosis induction [26], the complex picture of the giant differentiation as a process triggered and controlled (Exp. 4) by cell contacts is not at all an unexpected result. To account for the many different results obtained concerning giant formation in O. bifaria, we suggest the existence of what we called a "Labile Memory Counter" (LMC). Such a LMC would be/is a cellular device capable (a) of counting the cell contacts activating cortical keys, (b) of recording them for a certain time lag (labile memory) and (c) of adding the new contacts to the total: when the sum reaches a certain value (over a certain period of time?) giant differentiation is triggered. Whatever the fine, molecular nature of such a LMC, it seems to account for many characters of the process. First of all the existence of strain-specific thresholds differing from one another (different strains form 1 giant at different cell densities) can be now interpreted according to the LMC working hypothesis as due to one, or more, of the following factors: (a) a different strain specific number of cortical activating keys per surface unit, (b) a different strain specific number of contacts, sufficient for the induction to occur, (c) a different forgetting velocity, (d) a different key activation energy. The last hypothesis, in particular, seems well supported by the results of Exp. 1 here described: killed cells (= "still" cells) cannot induce any differentiation of gigantic cells, because the relative "bumps" are not sufficiently strong to activate the cortical keys. Also the results of Exp. 2 seem to fit perfectly into the LMC hypothesis. S 9 (which was known to produce giants more easily than S 6) shows both a lower threshold and a shorter time lag to produce the first giant than S 6 (Fig. 1). It was already known [28] that the induction of giant formation in O. bifaria is not species-specific (Paramecium proved to be capable of inducing it quite efficiently). The results here reported about B. japonicum as giant inducer in o. bifaria, beyond confirming the previous results, demonstrated a new, quite surprising point: not only can Oxytricha be induced to differentiate giants feeding on non-conspecific preys (Paramecium) but it can

also be pre-activated by cells (B. japonicum) which do not lend themselves as preys. These two complementary findings show that the keys of the LMC are neither species specific nor, more exactly, prey-specific. The experiments of Ricci et al. [28] showed that Con-A can specifically inhibit the induction of giant formation: according to a very wide literature [1, 16, 34, 35] this peculiar effect of Con-A strongly indicates that glycoprotein components (at least) are involved in the key-system, which is rather "aspecific", resembling a single "finger-and-key-system", rather than the highly specific lock-and-key one. To conclude our remarks about the LMC hypothesis, let us recall the results of Experiment 4 which treated both the giants and the preys with Con-A: already formed giants need a constant influx of input even to maintain their differentiation. This trait of the giant's biology seems to fit perfectly into the interpretation of this differentiated state of o. bifaria as an opportunistic shift from the normal feeding niche of this species (primary consumer) to a new one (secondary consumer) to extend its own temporal presence in a certain microhabitat [28]. The continuous control of the differentiation maintenance, by a LMC system, would enable the species to exploit the potential environmental sources (contacting preys) as long as they are sensed (perceived): soon after their disappearance (changed environmental conditions), the giants de-differentiate to normal cells (through 3 successive peculiar binary fissions) [27], spreading through the environment, in the search for new favourable conditions for a flourishing population (high bacterial density). According to the observations of Corliss [2] on the biology of the "tomites" of Tetrahymena and to the interpretation of their adaptive significance given by Fenchel [5], we are presently studying the behavior of the ex-giants of o. bifaria in an attempt to obtain actual measurements [23] possibly supporting the point of view according to which the locomotion of these cells should enable the species to disperse through the environment (searching for new microsources of food) with a greater efficiency than normal cells. Experiment 3 (magnetic stirring and giant differentiation) and Experiment 4 (B. japonicum and giant differentiation) yielded a second, relevant "fruit", namely the demonstration that the biological events occurring during the induction period are actually formed by at least two distinct successive steps: (1) the activation, which is cell contact dependent and which corresponds to the time lag necessary for the LMC to reach the proper threshold to induce a s.l. internal readiness and (II) the actual giant cell differentiation, which depends upon the encounter and the engulfment by an activated cell of a certain number of preys. Part of the biological events leading to activation will be discussed in the next paper [29]. Now, we should like to stress the consistence of the different informations gained by the round of experiments here described, which suggest the general traits of the two step mechanism leading to the formation of the carnivorous giants of O. bifaria. Such an extremely opportunistic nature of these peculiar forms (according to their interpretation given by Ricci et al.) [28] well accounts for the absolutely aspecific nature of both the input-key-system of the LMC (every

132 . N. Ricci, G. Grandini, A. Bravi and R. Banchetti

living object contacting properly an oxytricha can elicit a certain degree of activation, even if it does not lend itself as a prey, as B. japonicum) and of the predatory behavior (every living object, within well defined size ranges, not B. [aponicum, represents a potential prey). As a sort of final remark, some words must be spent to account for the choice of the term "cell differentiation", in relation to the heteromorphic phenomena of a ciliate: although the term per se has been purloined from developmental biology, the extrapolation seems to us quite appropriate, on the basis of the observation that both shape and physiology of each ciliate, living in one of these differentiated states, are changed according to a perfectly encoded pathway [30,32]. Two differences seem to distinguish the case of protozoa from that of metazoa: (a) the unicellular nature of the protozoan body cannot allow any irreversibility of the process, unless its very adaptive significance (to match internal and external conditions) is vanified; (b) while the differentiative adaptive answer of any protozoon cannot but be induced by external triggering conditions, the metazoan cell differentiation at the basis of individual development is a "built-in", self triggered and controlled process.

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Key words: Oxytricha bifaria - Giants - Differentiation - Cell-cell contacts Nicola Ricci, Dipartimento di Scienze dell'Ambiente e del Territorio, via A. Volta, 4, 56100 Pisa, Italy