Cell Size and Proportional Distance Assessment During Determination of Organelle Position in the Cortex of the Ciliate Tetrahymena

J. Cell Set. ai, 35-46 (1976) 35 Printed in Great Britain

CELL SIZE AND PROPORTIONAL DISTANCE ASSESSMENT DURING DETERMINATION OF ORGANELLE POSITION IN THE CORTEX OF THE CILIATE TETRAHYMENA

D. H. LYNN AND J. B. TUCKER Department of Zoology, The University, St Andrews, Fife KY16 <)TS, Scotland

SUMMARY Developing oral organelles of dividing Tetrahymena corlissi appear to be positioned by mechanisms which assess distances as a proportion of the organism's overall dimensions. In some respects, the cortex of this protozoan obeys the 'French flag' rule formulated by Wolpert for describing regulation of spatial proportions during differentiation of metazoan embryos. Dividing Tetrahymena of markedly different sizes occur when division is synchronized by starvation and refeeding. At the start of cell division, the distance between old and new mouthparts varies proportionately with respect to cell length. In addition, determination of the site where new oral organelles will develop is apparently not directly related to the number of ciliated basal bodies which separate the 2 sets of mouthparts; the greater the distance between the old and developing sets of mouthparts, the greater the number of ciliated basal bodies in the rows between them. It is suggested that 2 distinct mechanisms are largely responsible for defining organelle position in ciliates. The new terms structural positioning and chemical signalling are denned to describe these mechanisms.

INTRODUCTION Organelles are positioned in a very precise and specific fashion in many unicellular organisms. Precisely positioned organelles form particularly well ordered and charac- teristic patterns in the cortices of ciliates. The spatial complexity of these organelle arrays is comparable with the arrangement of different cell types, tissues, and organs in multicellular animals. Moreover, the body sizes of ciliates and the sizes of positional fields in multicellular animals and their embryos are quite similar (Frankel, 1974)- This fact, together with others, has led Frankel (1974) to argue that some of the mechanisms underlying the spatial specification of pattern are the same in both ciliates and multicellular animals. Universality of mechanisms for specifying positional information, particularly among metazoa, has been proposed in Wolpert's (1969, 1971) theory of 'positional information'. Wolpert has emphasized the fact that positional fields are regulative. Regulative fields are capable of proportionately re- establishing the same pattern after their boundaries have been disturbed or altered. Wolpert (1969) has formalized this in his 'French flag' rule. Examples of such regulation are well documented for metazoan development (see Wolpert, 1969; Cooke, 1975)- 3-2 36 D.H. Lynn andj. B. Tucker Strict proportional regulation has not been clearly established for ciliates. Frankel (1974) has reviewed microsurgical experiments which demonstrate that Stentor obeys the 'French flag' rule within certain limitations. There have been several morphometric analyses which demonstrate that positioning of contractile vacuole pores in Tetrahymena (Nanney, 1966, 1967; Frankel, 1972; Doerder, Frankel, Jenkins & De Bault, 1975) and Chilodonella (Kaczanowska, 1974) is probably accomplished by assessment of the overall size of the positional field in which they develop. Kac- zanowska (1974) and Jerka-Dziadosz (1974) have discussed the congruence between the models of positional information for multicellular and unicellular organisms. Many new cortical organelles develop when a ciliate commences binary fission. Is spacing of the sites where new organelles form proportionately related to the sizes of dividing organisms ? It is difficult to assess whether such spatial regulation occurs, rather than positional specification by mechanisms relying on fixed or absolute distances, because, before starting to divide, most ciliates reach a specific, well defined size which shows little variation. However, these alternatives can be tested with the hymenostome Tetrahymena corlissi. In this species, dividing organisms of markedly different sizes occur during refeeding following a period of starvation. Little growth of the organisms takes place in the unusually short period which intervenes between the 2 fissions which follow release from starvation (Lynn, 1975). This paper examines the extent of spatial proportionality in the cortex of T. corlissi, by measuring the distances separating the sites of old and differentiating new oral organelles at the start of binary fission in organisms of varying lengths. Differences in the number of cortical fibre-lattice units situated between the 2 sets of oral organelles have also been investigated. The results are discussed in terms of recent proposals for positional determination in metazoans (Wolpert, 1969, 1971) and in ciliated protozoa (Frankel, 1974, 1975; Sonneborn, 1974).

MATERIALS AND METHODS Culture techniques Tetrahymena corlissi strain WT, clone TC-2, was cultured axenically in 2 % (w/v) proteose- peptone with either o-i % yeast extract or o-i % neutralized liver digest. Dividing organisms of markedly different sizes are present after starved organisms are resupplied with nutrient culture medium. A variation of the starvation-refeeding technique described by Cameron & Jeter(i97o) was employed. A 200-ml culture of logarithmically growing T. corlissi (ca. 7000 cells/ml) was centrifuged to concentrate the organisms which were then washed twice in an inorganic 'starvation' buffer (Cameron & Jeter, 1970) and resuspended in 200 ml of starvation buffer. After 24 h, starved organisms were centrifuged down and resuspended in nutrient culture medium. The time at which the organisms started to divide again after release from starvation was ascertained by taking small samples from a culture at regular intervals, fixing the organisms in Lugol's iodine, and counting the numbers of dividing organisms in a standard volume. Experiments were conducted at 18-20 °C. Tetraliymena pyriformis strain W was cultured axenically in 1 % (w/v) proteose-peptone and o-i % yeast extract. Proportional distance assessment in Tetrahymena 37

Staining and microscopy The cortex of dividing organisms was stained with silver (Chatton & Lwoff, 1930; Corliss, 1953), protargol (McCoy, 1974) and nigrosin (MacKinnon & Hawes, 1961). Organisms were photographed with a Carl Zeiss Universal microscope fitted with bright-field, phase-contrast and Nomarski differential interference-contrast optics. Silver-stained organisms were measured with a Leitz filar ocular micrometer mounted on a Leitz Ortholux microscope.

RESULTS Positioning of new mouthparts At the beginning of binary fission each organism develops one new set of mouthparts. These form some distance from, and posterior to, the pre-existing old mouthparts (Fig. 1). They usually develop alongside a ciliary row (kinety 1) which extends

30 fim

Anterior mouthparts

Kinety 1

Developing posterior mouthparts

Fig. 1. Schematic scale drawing of first (I) and second (II) post-starvation dividers based on average measurements for 50 silver-stained specimens of each type of divider. d is the distance between mouthparts and / is the body length. The number of black dots in the first kinety of each divider represents the average number of basal bodies in the portion between the mouthparts. The old anterior mouthparts each include a curved undulating membrane and 3 membranelles. posteriorly along the length of the organism from the posterior end of the old set of mouthparts (Fig. 1). The distance (d) between old and new mouthparts (measured from the posterior end of the undulating membrane of the old anterior set of mouthparts to the anterior extremity of the developing posterior set of mouthparts) has been measured for silver-stained organisms at an early stage of binary fission and stomatogenesis (Fig. 1). By this stage, the basal bodies of the developing oral ciliary organelles form a compact group but have not sorted out into the distinct arrays D.H. Lynn and J. B. Tucker Proportional distance assessment in Tetrahymena 39 which will form the undulating membrane and 3 membranelles (Fig. 1). The lengths of dividing organisms in logarithmically growing cultures {log dividers) vary between 6o-8 and 77-6/tm and their mouthparts are separated by distances of 14-1-21-8/un (Table 1). Much greater variation in these parameters is found in organisms after release from starvation.

300

.:> .* o. 200

100

00 400 500 600 70 0 800 Body length, Fig. 2. Graph showing the relationship between the distance between the mouthparts (d) and body length (/) for 150 silver-stained dividing organisms (50 each of first and second post-starvation dividers and 50 log dividers). The line fitted by linear regression analysis has the equation d = o-3O55(/) - 2'474, where d and / are in /Jm.

After transfer to the starvation buffer, the total number of organisms increased by about 10% over 24 h. The sizes of the organisms decreased as starvation pro- ceeded. After 24 h, the lengths of organisms averaged 53-3 fim (43-3-61-5/tm; N = 15) while the widths averaged 18-2 /tm (14-7-22-5 /tm; N = 15). Non-dividing organisms in logarithmically growing cultures have lengths averaging 64-6 /tm (51-1-75-6/4111; N = 15) and widths averaging 32-9fim (25-2-39-0fim; N = 15). Organisms start to divide again about 12-15 n a^-er transfer from the starvation buffer to a nutrient culture medium (see Materials and methods). During this 3-h period, division synchrony of up to 10% was achieved by the starvation-refeeding procedure. This included organisms dividing for the first {first dividers) and second time {second dividers) after starvation. Further divisions do not occur for several hours after these 2 divisions have been completed. The second division usually begins within 1 h of completion of the first division. This is an unusually brief interfission period as these organisms have a generation time of about 26 h in cultures which are growing logarithmically. First dividers are about the same size as log dividers (Table 1). Second D. H. Lynn andjf. B. Tucker Proportional distance assessment in Tetrahymena 41 dividers are much smaller than first dividers (Figs. 1,3) or log dividers (Table 1). The lengths (/) of first dividers vary between 65-0 and 79-4/tm; their lengths do not overlap those of second dividers which lie in the range 44-0 to 61-7 /tm. Correlated with this, the distance (d) between the mouthparts is greater for first dividers than it is for second dividers (Table 1). The length range of log dividers overlaps the length ranges of first and second post-starvation dividers (Table 1). Comparison of the ratio d/1 (see Fig. 1) for the 3 types of dividers reveals that a fairly precise proportionality is maintained between d and / in organisms of different size (Table 1). Correlation analysis of d and / yielded product-moment correlation coefficients, r = 0-52 for log dividers, r = 0-43 for first dividers, and r = 0-57 for second dividers. These coefficients are significant at P = o-oi with 48 degrees of freedom. Moreover, a scatter diagram and linear regression analysis reveal that d and / are proportionately related when they are compared for dividers from log cultures and post-starvation cultures, whose lengths vary from 44-0 to 79-4 /im (Fig. 2). However, regression analysis indicates that d and / are not related in an exactly proportional fashion. Regression of djl on / gives djl = o-ooo663(/) + 0-2234. ^n tn^s equation, the slope is significantly different from zero at P = 0-05. Hence, djl increases slightly with /. Although proportionality is maintained between d and /, the length of the region occupied by oral basal bodies of new mouthparts is significantly greater in second dividers than it is in first dividers (Fig. 1) or log dividers. In addition, the lengths of old mouthparts are significantly smaller in second dividers than they are in first dividers (Fig. 1). The reasons for these differences have not been established.

Number of ciliated basal bodies between mouthparts The number of cilia in the portion of kinety 1 which extends between the 2 sets of mouthparts in dividing organisms of different lengths has been counted at the stage shown in Fig. 1. Tetrahymena pyriformis possesses cortical extrusion bodies called mucocysts; electron microscopy has revealed that they are distributed both between and within ciliary rows (Allen, 1967; Satir, Schooley & Satir, 1973). In T. corlissi, mucocysts are stained by both the silver and protargol procedures. The resulting 'silver dots', at or near the cell surface, have a similar appearance to those which indicate the positions of cilia and their basal bodies. Many mucocysts lie in the cortical zones between ciliary rows (Figs. 4, 5). As in T. pyriformis, some may also occur within ciliary rows. If this is the case, the number of 'silver dots' in a kinety will be

Fig. 3. Late furrowing stages of a post-starvation first divider and a much smaller second divider. Living Tetrahymena corlissi. Nomarski differential interference- contrast. Figs. 4, 5. Portions of the cortex of T. corlissi after Chatton-Lwoff silver-staining. The ciliary rows are oriented approximately parallel to the sides of the micrographs. All the silver dots between rows show the positions of mucocysts; some of the dots within the rows may also do so. x 3000. Fig. 6. Portion of the cortex of T. corlissi which has been negatively stained with nigrosin. Each circular black deposit represents a relatively large accumulation of stain which fills the cortical depression at the base of a cilium. x 3000. 42 D. H. Lynn andj. B. Tucker greater than the number of cilia. Because of this uncertainty, ciliary number has been estimated for organisms which were negatively stained with nigrosin. Stain collects in the cortical depressions where the tops of basal bodies are situated. The cilia are also apparent (Fig. 6). Mucocysts are not revealed by this staining procedure. The number of silver dots in the portion of kinety i, which separates the 2 sets of mouthparts in each type of divider, is on average only slightly greater than the number of cilia revealed by nigrosin staining (Table i). The number of cilia varies consider- ably in organisms of different lengths. More ciliated basal bodies are present in longer organisms than in shorter ones (Fig. i, Table i). The post-starvation first dividers can be distinguished from log dividers although similar distances separate the mouthparts in both types of dividers. Post-starvation first dividers have more basal bodies between the mouthparts than log dividers. The basal bodies of first dividers are less widely spaced than those of log dividers and second dividers (Table i). Protargol staining of T. pyriformis reveals that approximately 17 % of basal bodies in ciliary rows do not bear cilia during early stomatogenesis (Nanney, 1975). The large number of mucocysts stained by protargol in T. corlissi prevents such an assessment for this species. In T. pyriformis there is certainly variation in the total number of basal bodies (ciliated and non-ciliated) between developing mouthparts; examination of 32 protargol-stained log dividers revealed that the number ranged between 9 and 17 (mean = 12). As in T. corlissi, longer organisms generally have more basal bodies between the mouthparts than shorter ones.

DISCUSSION Structural positioning and chemical signalling Two distinct types of positional mechanisms may be responsible for determining spatial differentiation in ciliates. The nature of these 2 mechanisms is outlined below. The somatic cortex of most ciliates possesses a complex, repeating, subpellicular fibre-lattice which often includes microtubules, microfilaments, and striated ciliary rootlet fibres. Most of the remaining cortical organelles, such as somatic cilia, contractile vacuole pores, and oral organelles, are structurally associated with this fibre- lattice. Determination of the position and spacing of developing organelles may be effected by nucleation of their assembly by particular sites on pre-existing elements of the fibre-lattice. Growth or contraction of contiguous elements in the lattice may also be responsible for arrangement of the organelles within it. Such mechanisms involve procedures for which the general term structural positioning is suggested. Structural positioning refers to instances in which structural contact with a pre- existing structure is necessary during definition of the position of a new or developing structure. For example, new basal bodies often start to assemble in contact with, and at a precise orientation to, mature basal bodies {Dippell, 1968; Allen, 1969; Millecchia & Rudzinska, 1970). Moreover, structural and genetic analysis of paramecia with inverted ciliary rows leaves no doubt that the orientation of pre-existing structures in the fibre-lattice influences the arrangement of new organelles which develop in close proximity to them (Beisson & Sonneborn, 1965). Proportional distance assessment in Tetrahymena 43 Alternatively, determination of the relative positions of organelles may depend on the same sort of mechanisms as those which define the sizes and positions of tissues during metazoan embryogenesis, particularly where spacings of several microns are concerned (Frankel, 1974). Such mechanisms may depend on spatial variation in the concentration of certain chemicals, possibly achieved either by diffusion (Wolpert, 1969; Crick, 1970) or by electrochemical activity of cell surface membranes (Frankel, 1975). The general term chemical signalling is proposed for instances in which the positions of new structures are determined by spatial differences in the concentrations of chemicals which are not tightly bound to new or pre-existing structures. Chemical signalling, unlike structural positioning, does not rely on interactions between contiguous elements of a cytoskeleton. Frankel (1974) has distinguished 2 modes for definition of organelle position in ciliates. One mode, structural guidance, operates over short distances. The other is a long-range mode which may be based on chemical signalling. Pre-existing structures define the positions of closely adjacent new structures during structural guidance, although, unlike structural positioning, direct structural contact may not always be involved.

Absolute or proportional distance assessment? The results presented above demonstrate that in T. corlissi, the site at which new oral organelles develop is determined by mechanisms which take into account the overall length of the organism. The distance separating the mouthparts varies as a proportion of the length of an organism. New mouthparts do not develop at a point which is separated from the old mouthparts, the poles of the cell, or any other obvious reference point, by a fixed or absolute distance. While structural positioning may define the spacing of adjacent organelles in terms of fixed invariant distances, for example the spacing of adjacent membranelle basal bodies in Nassula (Tucker, 1971), it is much more difficult to see how structural positioning can establish proportionality of organelle spacing with respect to cell size. On the other hand, the ways in which spatial proportionality might be regulated by positional mechanisms based on chemical signalling are well defined in theory, particularly for model mechanisms in which signalling is based on a concentration gradient of a diffusible chemical (Lawrence, 1966; Wolpert, 1969; Crick, 1970; Lawrence, Crick & Munro, 1972; Jerka-Dziadosz, I074)- Spacing of mouthparts in T. corlissi might be determined by an absolute mechanism in which a particular number of cortical fibre-lattice units separates the old mouthparts from the site at which new ones form when the position of the latter is determined. In Tetrahymena, each such unit, or cortical territory, is usually associated with a single mature basal body and its cilium. However, the number of cilia in the portion of kinety 1 between the mouthparts varies in dividing organisms. Longer organisms have more somatic cilia between mouthparts than shorter ones. Thus, the site of oral morphogenesis is apparently not defined by any absolute mechanism, even one which 'counts' a fixed number of cortical units. This conclusion is based on the assumption that the site is determined shortly before organisms reach the stage at which measure- 44 D. H. Lynn andj. B. Tucker ments have been made (Fig. i). The site may be determined much earlier in the cell cycle, before developing mouthparts can be detected, when the organism is shorter and perhaps has fewer cilia and cortical units. If this is the case, site determination might still be based on a mechanism which defines an absolute distance or a fixed number of cortical units. At this earlier time, the distance between the 2 sets of mouthparts may be a certain proportion of body length since the organism might determine the site either when it has grown to a particular length, or when kinety 1 includes a particular number of cilia, assuming that cilia and their basal bodies are more or less evenly spaced along the kinety. In either case, this spatial proportionality must be maintained from this early time of determination of oral position until organisms reach the stage at which measurements were made. Uniform growth throughout the cortex would be the easiest method of maintaining the proportionality of distance, between the old mouthparts and the new oral site, relative to the organism's length. It seems unlikely that the new oral site should be determined at varying times in the cell cycle when the organism has grown to a precisely specified length or possesses a specific number of ciliated basal bodies in kinety 1. Hence it is probably correct to assume that the position of the site is determined by a mechanism which defines distances between mouthparts in terms which are related proportionately, rather than absolutely, to cell length.

Migratory and in situ cortical organelle morphogenesis The new oral organelles of Tetrahymena develop in situ. At the start of their development they are situated in the position they will finally occupy relative to other cortical regions. For certain cortical organelles in other ciliates, the definition of position is apparently more complex. In some cases, new organelles may form close to old ones initially. Then, as development progresses, the new organelles migrate away from the old ones for several microns through the cortex, travelling past adjacent cortical regions not included in the migration. Increases in the distances separating such organelles are sometimes due partly to migration and partly to a general expansion of the cortex associated with growth of the organism (Tucker, 1971). This expansion is often quite rapid during and after fission. The new mouthparts of Paramecium, and several other ciliate genera, are initially situated close to the old ones. The 2 sets of mouthparts become more distantly separated as division pro- ceeds (Hanson, 1962; Sonneborn, 1963). The new cirri of some hypotrichs start to form in close association with each other and subsequently move apart (Grimes, 1972; Jerka-Dziadosz, 1974). In such instances, structural positioning may be involved in defining the site where new organelles initially start to form. A particular part of a pre-existing organelle, or part of the cortical fibre-lattice, may nucleate assembly of a new organelle. Structural positioning may also be involved in migration of organelles to their new positions. For example, in Nassula, bundles of microtubules and microfilaments run between migrating contractile vacuole pores and the cortical regions which they are approaching (Tucker, 1971). Nevertheless, the possibility remains that the destinations of migrating organelles are defined by chemical signals. Regulation of the distance migrated would occur by Proportional distance assessment in Tetrahymena 45 proportional assessment of the overall dimensions of an organism. However, one wonders why migrations occur if ciliates really can map out a proportionately regulated pattern of regional differentiation by means of chemical signals. It would seem simpler for the organelles to form in situ. When organelles migrate, their destinations may be determined entirely by structural positioning. Proportional regulation of spacing with respect to cell size could be achieved by structural positioning, if larger organisms synthesized more fibre precursors than smaller ones. The lengths of these fibres, which guide, push, or pull developing organelles to their final positions, might then be proportionately related to the size of the organism.

Post-starvation divisions Starvation-refeeding treatment may interfere with the control of the normal se- quence and number of events which precede binary fission. In logarithmically growing cultures, only one size class of divider was encountered (Table 1), while there are usually 2 size classes of dividers in post-starvation cultures. The larger divider in post-starvation cultures, the first divider, possesses more cilia per unit length of kinety and is larger than the log divider (Table 1). Thus, first dividers may possess more cortical organelles and contain greater pools of precursor materials than is normally the case. In first dividers, the quantity of certain precursors whose concentration triggers binary fission may be about twice the necessary quantity so that daughter organisms (i.e. second dividers), although much smaller than normal (Table 1), are 'ready' for division without delaying for further syntheses. This may be the reason why the second post-starvation division follows the first after an abnormally brief interval and before much interfission growth has occurred. We thank Dr J. Frankel for critically reading the manuscript and Mr C. D. Sinclair for his statistical advice. This work has been supported by grants B/SR/88418 and B/SR/5894.5 from the Science Research Council (U.K.). D.H.L. acknowledges a NATO Postdoctorate Fellow- ship awarded by the National Research Council of Canada.

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