DIMINUTION OF MICROTUBULAR ORGANELLES AFTER EXPERIMENTAL REDUCTION IN CELL SIZE IN THE CILIATE, DILEPTUS

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1 J. Cell Sci. 70, (1984) 25 Printed in Great Britain The Company of Biologists Limited 1984 DIMINUTION OF MICROTUBULAR ORGANELLES AFTER EXPERIMENTAL REDUCTION IN CELL SIZE IN THE CILIATE, DILEPTUS KRYSTYNA GOLINSKA Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw , Poland SUMMARY Organelles of two kinds were studied: nemadesmata and transverse fibres, which are kinetosomal rootlets in oral parts of Dileptus. A diminution of microtubule number and length was found in organelles of size-reduced and regenerating cells, as compared to normal ones. It appeared, however, that organelles reduced in size were always larger and longer than could be expected on a basis of perfectly proportional regulation. The results give some insight into the modes of formation and regulation of the size of these organelles. INTRODUCTION The elaborate cortical pattern of ciliates has repeatedly been found to be sufficiently flexible to fit abnormally small or large cells. Compensatory change in size-reduced ciliates has been found in both the number of multikinetosomal ciliary complexes and the number of kinetosomes within these complexes (Jerka-Dziadosz, 1976, 1977; Bakowska & Jerka-Dziadosz, 1980; Bakowska, 1980, 1981; Bakowska, Frankel & Nelsen, 1982). The number of microtubules forming the kinetosomes was found to be unaltered. In this study a reduction in microtubule number and length was observed in single organelles, when very small cells were compared with normal ones. The organelles studied were root fibres of oral kinetosomes: namely, nemadesmata and transverse fibres in Dileptus. In ciliates other than Dileptus it has been found that the nemadesmata, hexagonally packed bundles of microtubules, both form and grow during stomatogenesis. A tiny bundle of several microtubules isfirstobserved, after the formation of which the nemadesma enlarges by lateral addition of microtubules (Paulin & Bussey, 1971; Pearson & Tucker, 1977). The lengths of nemadesmata may change not only during the stomatogenic period: starvation and refeeding cause shortening and elongation of these organelles in Nassula (Tucker, 1970). It remains unclear whether the size of a nemadesma, i.e. the number of its constituent microtubules, can be altered in mature mouthparts. The transverse fibres, the other kind of organelle analysed in this study, are long ribbons of microtubules situated under the cell surface and connected to the proximal end of a kinetosome. Nothing is known about the capacity of ciliates to alter the number and length of microtubules in their transverse fibres. 2 CEL 70

2 26 K. Golinska The aim of this study was to ascertain whether reduction in cell size leads to reduction in the number and length of microtubules in kinetosomal rootlets, i.e. whether the mechanism controlling regulation of ciliary pattern may work at the level of a single organelle. It was found that downward regulation of microtubule number and length occurs in root fibres, although it is not proportional in extent to the reduction in cell size: the organelles are always larger and longer than would be predicted on the basis of perfectly proportional regulation. MATERIALS AND METHODS Dileptus anser, a gymnostome ciliate, was used in this investigation. Culture methods were described in detail in a previous study (Golinska & Jerka-Dziadosz, 1973). Cells were reduced in size by transactions. The operations were performed by hand, using a microknife. The length of cells was measured using specimens fixed with a saturated aqueous solution of mercuric chloride, then washed and observed in water. For light-microscope observations protargol staining was carried out, using the slightly modified method of Tuffrau (1967). The following modifications were introduced: (1) The fixative used was a mixture of 10 parts of saturated aqueous mercuric chloride and one part of 1 M-HC1. (2) After treatment with sodium hypochlorite, specimens were rinsed with a 1 % aqueous solution of tannic acid, then immersed in the same solution for 15-3Omin. With this technique both surface fibrous structures and deep fibres, such as the nemadesmata of the pharyngeal basket, are stained. Material for electron microscopy was prepared as in a previous study (Golinska, 1982), and examined in a JEM 100 B transmission electron microscope. Size-reduced cells only were prepared especially for this study. The micrographs of regenerating and normal cells were made during previous studies of D. anser. For each group of cells data were taken from more than 20 specimens. RESULTS The organelles studied are microtubular derivatives of non-ciliated kinetosomes in the mouthparts of Dileptus. The fine structure of Dileptus has been described previously (Grain & Golinska, 1969; Golinska & Kink, 1976), and a brief description only is needed here. Dileptus consists of an elongated cylindrical trunk, with a slender process at its anterior end called the proboscis (Fig. 1A). The cytostome is situated at the base of the proboscis, in the middle of a circular cytostomal field. A narrow extension of this field occupies the ventral side of the proboscis along its whole length, and is called the ventral band. On the margin of the cytostomal field and the ventral band there is a continuous row of cilia forming the oral ciliature (Fig. 1B). Kinetosomes of this row give rise to both nemadesmata and transverse fibres. The part of the oral ciliature that encircles the cytostome consists of onekinetosomal elements, each equipped with nemadesma and transverse fibre. The number of microtubules was analysed in both nemadesmata and ribbons of transverse fibres. On both margins of the ventral band there are rows of kinetosomal pairs. The kinetosome on the outer side of the ventral band bears a cilium. That on the inner side is non-ciliated and bears a transverse fibre directed towards the middle of the band (Fig. 1B). The fibres coming from the right side are intermingled with trichocysts

3 Diminution of organelles in small cells 27 Fig. 1. A. Ventral view of Dileptus. The dotted area is the cytostomal field, B. Pattern of transverse fibres in the cytostomal field. 1, Right transverse fibre; 2, left transverse fibre; 3, transverse fibre of cytostomal area; 4, nemadesma. (Fig. 2). The transverse fibres coming from the left side of the ventral band run only a short way toward the middle of the band, then curve and run posteriorly, in the direction of the cytostome. These curved parts of the transverse fibres form a bundle in the territory of the ventral band (Figs 1B, 6, 7), the so-called central fibre. Microtubules were counted in transverse fibres on both the left and right sides of the ventral band. Moreover, the microtubular ribbons in a given section of central fibre were counted, in order to estimate the length of left transverse fibres. The organelles studied originate from three groups of cells of different sizes. To estimate the size of cells, only the length of their trunks was measured, from the base of the proboscis to the end of the tail (Fig. 1A). This is justified by the observation that there is the proportional regulation of size of cell parts in Dileptus (Golinska, 1979). The largest cells studied were those taken from growing culture, in all possible stages of the cell cycle except late fission. These cells were termed normal cells, and their trunk was on average 319/xm long (Table 2). The smallest were 'size-reduced' cells, obtained by cutting Dileptus into tiny fragments, and leaving them for a day to regenerate. No food was given to these animals, in order to obtain very.small cells. The average length of their trunks 24 h after the operation was 109 fxm. Thus there

4 K. Golinska Figs 2-4

5 Diminution of organelles in small cells 29 was a threefold reduction of the cell length in size-reduced animals in comparison to normal ones. A third group of cells are the posterior fragments 2-4 h after the operation, which have newly formed nemadesmata and transverse fibres. At this stage of regeneration fragments contain complete mouthparts, although not all of them are ready to function (young mouthparts were observed to start swallowing 3h after the operation; Golinska & Bohatier, 1975), and they are still growing. Since at this stage the new oral organelles are of comparable size in small and large fragments, the length of fragments has not been taken into consideration. Analysis of microtubule number in nemadesmata Nemadesmata of Dileptus belong to the type that are permanently connected to kinetosomes (classification of organelles after Grain, 1969). Like such organelles in other ciliates, the nemadesmata of Dileptus are bundles of tightly packed and interconnected microtubules, held together in their proximal part by a matrix plate ('plaque matricielle' of Didier, 1970), and attached to the basal part of the kinetosome and its transverse fibre by electron-dense material (Figs 3,4). Nemadesmata form an almost complete circle around the cytostomal field (Fig. 1B), and the nemadesmal basket penetrates deeply into the cell (Fig. 2), encircling the inner ring of microtubular bundles, which make up the so-called internal basket (Figs 2, 8; for details see Golinska, 1978). Because of the very large number of nemadesmata in the outer basket, their crowding and the variability of their dimensions, it was not possible to count the total number of nemadesmata per basket. The data presented here concern only the size of nemadesmata. The size of a nemadesma was estimated by counting the microtubules making it up. The number of microtubules was counted in transverse sections taken close to the kinetosome. This was to avoid an error in estimation coming from the possibly unequal length of microtubules in a nemadesma. Nemadesmata of Dileptus have no defined shape. Usually roughly oval in cross-section, nemadesmata may also be rhomboidal, triangular or irregular in shape. The big ones are intermingled with tiny bundles, without any apparent pattern in the distribution of nemadesmata of different sizes (Fig. 4). Nemadesmata found in normal, size-reduced and regenerating cells differ in the mean number of microtubules of which they are composed. In normal cells the mean Fig. 2. Mouthparts of Dileptus. c, cytostome; n, nemadesma; ib, internal basket made out of numerous bundles of microtubules. In the upper part of micrograph the basal portion of the proboscis is visible. Protargol staining. X4000. Fig. 3. Oral kinetosomes (ok) with nemadesmata (n). At the top of each nemadesma there is a matrix plate (mp) and amorphous matter (am). X Fig. 4. Transverse section of nemadesmal basket, c, an arrow indicates the direction toward cytostome; n, nemadesma; ok, oral kinetosomes on top of nemadesma; nk, newly forming oral kinetosomes just outside the basket; Ik, kinetosomes of locomotor cilia. X

6 30 K. Golinska Table 1. Number of microtubules in nemadesmata from three groups of cells Group of cells Mean no. of microtubules per nemadesma Range of microtubule no. Mode Median No. of nemadesmata analysed Normal Size-reduced Regenerating number of microtubules in a single nemadesma was 86 (Table 1), in size-reduced cells it was 64 and in regenerating cells Thus it can be said that the average nemadesma in size-reduced cells is reduced by about 20 % in microtubule number as compared to nemadesma from normal cells, showing that there is a downward regulation in size. When regenerating and size-reduced cells are compared, it appears that cells in the latter have nemadesmata about twice as big as those in the former. This indicates that nemadesmata can grow after the end of stomatogenesis, even when there is no food intake (in the case of size-reduced cells). The chi-square test was used to check whether the differences in size of nemadesmata found in the three groups of cells were significant. Nemadesmata were arbitrarily divided into five classes: tiny nemadesmata (1 30 microtubules), small (31 60 microtubules), medium ( microtubules), large ( microtubules) and very large (more than 200 microtubules). A histogram of microtubule numbers in nemadesmata in the three groups of cells is shown in Fig. 5. The chi-square analysis showed that the distribution of these classes of nemadesmata is significantly different in the three kinds of cells. The difference between the nemadesmata in the three kinds of cell lies not only in the mean microtubule number but also in the frequency distribution of nemadesmata of various sizes. In normal cells the frequency distribution is highly asymmetrical, drawn out in the direction of small nemadesmata (Fig. 5). The modal value is almost three times smaller than the mean number of microtubules per nemadesma (Table 1). The frequency distribution of regenerating cells is similar, although less asymmetrical, while in size-reduced cells an almost symmetrical frequency distribution is found. The above observations strongly suggest that new nemadesmata are added to pharyngeal parts in regenerating and normal cells, while in size-reduced cells this addition is markedly slowed down. In size-reduced cells the medium-sized nemadesmata prevail, while in normal cells the medium-sized and large organelles are present in equal quantities, and a class of very large nemadesmata is found only in this group. This suggests that in size-reduced cells the growth of individual nemadesma is somewhat limited. When the group of regenerating cells is taken into consideration, it appears that their nemadesmata belong almost entirely to tiny and small classes, those of medium size are rare, and large nemadesmata can only occasionally be found (Fig. 5). This indicates both that there is a very quick growth of nemadesmata during regeneration, and that this growth continues after regeneration is finished.

7 Diminution oforganelles in small cells 31 7% 12-6% 2% 10-4% 22% 40-6% 45-2% < % % 28-7% % % 18-1% 1-30 Normal Sizereduced n=160 Regenerating n = 250 Microtubules Fig. 5. Percentages of very large, large, medium, small and tiny nemadesmata (indicated by numbers of microtubules) found in baskets of normal, size-reduced and regenerating cells of Dileptus. Analysis of microtubule number in transverse fibres In this study only the transverse fibres that belong to non-ciliated oral kinetosomes were taken into consideration, and no data were collected concerning transverse fibres of kinetosomes that bear locomotor cilia. The numbers of microtubules were counted in transverse fibres from three regions on the circumference of oral field: (1) the cytostomal field, where the fibres are linked to kinetosomes bearing nemadesmata, (2) the right and (3) the left sides of the ventral band in the proximal portion of the proboscis. The transverse fibres within a given region of an individual mouth are composed of very similar numbers of microtubules, in sharp contrast to the variability of the nemadesmata, described above. The transverse fibres seem to contain microtubules of almost equal length, and only in their most distal portion are some of the

8 K. Golinska Figs 6 8

9 Diminution of organelles in small cells 33 Table 2. Comparison of microtubule number and length in transverse fibres of cells of different size Group of cells Length of trunk (;Um) No. of microtubules in transverse fibres Left fibre Right fibre Cytostomal fibre No. of ribbons in central fibre Normal 319 ± ±2-0 w= ±1-1 «= ±1-9 w = ±2-0 w = 96 Size-reduced 109 ±21 «= ± ± ±1-0 Regenerating Not measured 16-6 ± ± ± ±1-6 w = 17 M = 36 «= 55 w = 14 Values are given ± S.D. The broken line separates the values that differ significantly from each other. microtubules shorter than their neighbours. This can be observed in transverse sections of the central fibre (Fig. 7): only those transverse fibres that are situated on the extreme margin of their territory (on the right side of the central fibre for left transverse fibres, and in the immediate vicinity of the central fibre for right transverse fibres) contain sometimes fewer microtubules than their neighbours. Ribbons situated in the cytostomal field are reduced in microtubule number in the region where they enter the cytostomal depression. The number of microtubules was not counted in these far ends of the transverse fibres. A comparison of microtubule number in transverse fibres of normal, size-reduced and regenerating cells is presented in Table 2. The transverse fibres found in the cytostomal field are comparable in size to those on the right side of the ventral band. The ribbons from the left side of the ventral band are always composed of more microtubules than any other ribbons in the mouthparts (Fig. 7, Table 2). The left transverse fibre in cells of normal size is composed of about 26 microtubules. The same fibre in size-reduced cells contains on average 21 microtubules, and is significantly smaller than in normal cells. Thus reduction in cell size leads to a reduction of 23 % in microtubule number. Still fewer microtubules, Fig. 6. The ventral band on the proboscis. On the right side (the left of the photograph) toxic trichocysts (t) are situated between right transverse ribbons (rt). On the left side (right of the micrograph) pairs of oral kinetosomes (ok) are connected to left transverse fibres (It). The curved segments of those fibres form the central fibre (cf) directed toward the cytostome. X Fig. 7. Transverse section of the central fibre (cf). Left transverse fibres (It) contain more microtubules than right transverse fibres (rt); t, toxic trichocysts;m, mucocyst. X Fig. 8. Section of cytostomal field in the region of the internal basket of microtubular bundles (ib); ct, transverse fibres of oral kinetosomes bearing nemadesmata. X34000.

10 34 K. Golinska about 17, were found in left transverse fibres of regenerating cells. The difference in microtubule number between regenerating and size-reduced cells is also highly significant. This indicates that in young mouthparts small left transverse fibres are produced. In older cells (the size-reduced group) much bigger ribbons can be found on the left side of the ventral band, but they never reach the microtubule number found in left transverse fibres of a normal cell. The transverse fibres in the right side of the ventral band contain about 15 microtubules in normal cells, 12 in size-reduced cells, and 12 in regenerating cells. It appears that the right transverse ribbon of normal cells contains significantly more microtubules than transverse ribbons in cells of the other groups, and there is no significant difference between the transverse fibres of size-reduced and regenerating cells. This indicates that in young mouthparts the right transverse fibres are already as big as those found in size-reduced cells. Since the left transverse fibres are smaller in regenerating cells than in size-reduced ones, somewhere between 4 and 24 h after the operation, the transverse fibres formed at the right-hand base of the ventral band are all alike, while larger transverse fibres are formed at the left-hand base of the ventral band (the structural units of the ventral band are formed at the basis of the proboscis; Golinska & Kink, 1976). Reduction of microtubule number in right transverse fibres of size-reduced cells, as compared to normal ones, is about 20%. The reduction of microtubule number in right transverse fibres is slightly lower than that observed in left transverse fibres. The transverse fibres situated in the cytostomal field contain about as many microtubules as the right transverse fibres, in all groups of cells studied. In cells of normal size the mean microtubule number in fibres in the cytostomal field is 16-5, in size-reduced cells 12 and in regenerating cells Again, as in right transverse fibres, the difference between mean values obtained for size-reduced and regenerating cells is not significant, while the difference between mean values for normal and regenerating (or size-reduced) cells is highly significant. The reduction of microtubule number in transverse fibres of the cytostomal field, when groups of normal and size-reduced cells are compared, is about 25 % the highest found for transverse fibres. The threefold reduction in cell length thus results in relatively low reduction of microtubule number within all kinds of transverse fibres. The data presented above allow one to compare microtubular organelles of two kinds: the nemadesmata and the transverse fibres. The most conspicuous difference is the variability in the size of organelles within the same mouthparts as well as within the same group of cells. The nemadesmata are shown to differ enormously in their microtubule number, while the number of microtubules within the observed classes of transverse fibre varies only slightly, both in fibres belonging to the same cell, and in those belonging to cells of the same group. Estimation of microtubule length in the left transverse fibres The left transverse fibres are derivatives of oral kinetosomes from the left margin of the ventral band, and their curved distal parts form the central fibre (Fig. 6). The number of ribbons that constitute the central fibre in a given section equals the

11 Diminution of organelles in small cells 35 number of kinetosomal pairs to which the ribbons belong. Preliminary observations showed that the distance between kinetosomal pairs on the left border of the ventral band is practically the same in normal, size-reduced and regenerating cells. Since material has been collected over many years, the distance between kinetosomal pairs was not measured directly on micrographs, to avoid possible errors arising from different electron-microscopic procedures. Instead, the ratio of the distance occupied by five kinetosomal pairs to the length of a kinetosomal pair in cross-section was used. The mean value for normal cells (n = 12) was 5-54 (s.d. ± 0-49) and for size-reduced cells (n = 5) 5-31 (s.d. ± 0-84). The difference between these means is not significant according to Student's Mest (a = 0-05). In regenerating cells the mean value for the distance between kinetosomal pairs is similar to that in other groups of cells (5 1) but the variability in the distance is much higher (s.d. ± 1-5). Again, the mean for regenerating cells does not differ significantly from those obtained for normal and size-reduced cells. Since the distance between kinetosomal pairs on the left margin of the ventral band does not change with the size of the cell, the difference in the number of transverse fibres within a central fibre reflects the difference in length of these fibres, at least in the length of their curved portion. Data concerning the number of transverse ribbons in the central fibre of normal, size-reduced and regenerating cells are presented in Table 2. It appears that normal cells have about 12 transverse ribbons in their central fibres, while in size-reduced cells only seven ribbons were found. This indicates that in size-reduced cells the lengths of microtubules decrease almost twofold, at least in the portion of the transverse ribbon that belongs to the central fibre. In some cases the lengths of the left transverse fibres can be measured directly in electron micrographs. In a normal cell ribbons 10 fim long have been found, in a size-reduced cell the length of such a fibre was 6 fim. Sections showing the whole length of the fibre were, however, too scarce to allow extensive comparative studies. The mean number of ribbons in central fibres of regenerating cells was 6-6, which is not significantly different from the 7 ribbons found in size-reduced cells. This indicates that in the left transverse fibres of regenerating cells there is no increase in length of microtubules up to 24 h after the operation, in spite of the fact that there is an increase in microtubule number. The lack of dependence of the number of microtubules on their length is further confirmed by the degree of their reduction in size-reduced cells in comparison to normal ones, described above: while the length of microtubules is reduced almost twofold, their number diminishes by some 20%. DISCUSSION Timing and localization offormation of microtubular organelles in Dileptus mouthparts In ciliates other than Dileptus, such as Tetrahymena or Paraurostyla, multikinetosomal complex organelles of mouthparts are formed, and are adjusted to the cell size, only during the brief period of stomatogenesis. Once stomatogenesis is complete, no new ciliary complexes are formed, and probably single organelles are neither added

12 36 K. Golinska to nor withdrawn from these complexes in mature cells (see reviews by Jerka- Dziadosz & Golinska, 1977; Bakowska et al. 1982). The regulation of the size of Dileptus mouthparts seems to be independent of the stomatogenic period. The oral kinetosomes with their nemadesmata and transverse fibres may form and disintegrate not only during stomatogenesis, but also in the mature and functioning mouth, whenever there is a need to enlarge or diminish the mouthparts. The formation and disintegration of structural elements proceeds in separate areas: formation can be found on the outer margin of the pharyngeal basket, resorption is normally restricted to the apical portion of the proboscis (Kink, 1976; Golinska & Kink, 1976, 1977; Golinska, 1978, 1979). Since both proliferation and resorption of structural elements have been found in nearly all the mouthparts studied, the suggestion has been made that a continuous exchange of structural elements exists in Dileptus mouthparts, and the regulation of the size of the mouth (i.e. of the number of its structural elements) is achieved through adjustment of the rate of proliferation and resorption (Golinska, 1979). The consequence of this manner of regulating the size of the mouthparts is that their constituent organelles are of different age. Proliferation occurs randomly at the circumference of the cytostomal field, and the new kinetosomes with their nemadesmata and transverse fibres are inserted between already existing kinetosomes. Thus, organelles of different age are mixed up together in the cytostomal field. The elongation of the ventral band results from growth at its base, where new structural elements are formed (Golinska & Kink, 1976). Thus in the ventral band the transverse fibres located close to each other are of similar age, and the further away from the cytostome these fibres are situated, the older they are. Formation and growth of nemadesmata and transverse fibres The number and organization of microtubules within organelles are supposedly controlled through the activity of a special structure, the so-called microtubule organizing centre (MTOC of Pickett-Heaps, 1969). The MTOC contains microtubule-nucleating elements, and the number and spatial arrangement of these elements determine both size and shape of an organelle (see review by Tucker, 1979). Both nemadesmata and transverse fibres of Dileptus have their MTOC connected to the basal parts of oral kinetosomes. Structurally, the MTOC is represented by electron-dense amorphic material, in which are embedded the matrix plate of nemadesma, the basal end of the transverse fibre and the proximal end of the kinetosome. The MTOC of nemadesmata seems to act continuously or in a sequence of periods of activity, adding nucleating sites laterally to already existing ones. A similar process was observed by Pearson & Tucker (1977) in growing nemadesmata oinassula. The growth of nemadesmata in Nassula is, however, restricted to the period of their formation, and the nemadesmata in a basket are all of a comparable size. Moreover, the mature organelles are structurally different from the growing ones (Tucker, 1970). In Dileptus there is no such thing as a structurally immature nemadesma. The only structural difference between nemadesmata is in the number of their constitutive

13 Diminution of organelles in small cells 37 microtubules, no matter how old they are. The data obtained in this study, especially the wide range in size of nemadesmata and the highly asymmetric frequency distribution of their microtubule content, indicate that not only is there a continuous addition of new microtubules to nemadesmata, but there is also a continuous addition of new nemadesmata to the pharyngeal basket. In size-reduced cells the frequency distribution of microtubule number in nemadesmata is almost symmetrical. This indicates that the rate of formation of new nemadesmata can be more severely reduced than the addition of new microtubules to already existing nemadesmata. The MTOC of transverse fibres, once formed, does not seem to change the number of its constitutive nucleating elements. This is indicated by the uniform size of transverse fibres in the cytostomal field. Since the organelles there are of different age and similar size, it has been assumed that the transverse fibres, once formed, do not enlarge further by addition of new microtubules. It is interesting that MTOCs that act in an extremely different way, such as those of nemadesmata and transverse fibres, can be situated so close to one another. Regulation of size of nemadesmata and transverse fibres It has been found in this study that the number of microtubules in organelles can be regulated according to the size of the cell. The relation between the size of the cell and the size of its organelles has to be maintained through MTOCs. This means that MTOCs of nemadesmata and transverse fibres of Dileptus are the cell-size sensing structures, and express this by changing the number of their microtubule-nucleating elements. Regulation of size of transverse fibres takes place a long time after the end of stomatogenesis. This is indicated by the fact that regenerating fragments of different size form transverse fibres of uniform size, without reference to the dimensions of the cell. The fibres formed during regeneration are of minimum size, the fibres formed by tiny and starving cells of the size-reduced group containing more or the same number of microtubules. Since in young mouthparts transverse fibres of minimum size are formed, and in older and sufficiently large cells the regulation of size of organelles takes place, the small transverse fibres are gradually replaced by larger ones during continuous exchange of structural elements within the mouthparts of Dileptus. The regulation of size of nemadesmata is not so obvious as the regulation of size of transverse fibres. The mean value for microtubule number in size-reduced cells is, however, reduced by 25 %, which is similar to the reduction in microtubule number found in transverse fibres. The size of nemadesmata probably depends upon the global time of activity of its MTOC; thus, the regulation of nemadesmal size could operate through restriction on the duration of MTOC activity. It is possible that the regulation of size of transverse fibres occurs in the same fashion, i.e. the formation of a large transverse fibre in a large mouth lasts longer than the formation of small fibres in a small mouth. Of course, this supposition needs experimental verification. What informs the MTOC about the size of the cell, thus determining the number of their nucleating elements and the size of organelles, remains completely obscure. This unknown factor must, however, be present in the area where new transverse fibres and nemadesmata are forming, i.e. on the outer margin of the cytostomal field.

14 38 K. Golinska The length of microtubules and its regulation in organelles In size-reduced cells, in comparison to normal ones, there is an almost twofold decrease in the length of the left transverse fibres. Thus, the reduction in microtubule length (44%) is much more drastic than the reduction in microtubule number (25 %). The question arises as to whether the length of microtubules can be regulated through the same mechanism that decides on the number of microtubules. The length of microtubules in nemadesmata has been observed to change during starvation and refeeding in Nassula (Tucker, 1970). During different morphogenetic processes the ciliary shafts of some cilia undergo shortening and subsequent elongation (Ruffolo & Frankel, 1972; Hammersmith, 1976; Sawyer & Jenkins, 1977). Thus the length of microtubular organelles can change without a change in microtubule number. It is not known whether the length of microtubules is regulated through their MTOCs or through a shifting of the balance between formation and dissolution in their environment (see reviews by Raff, 1979; Tucker, 1979). Brinkley et al. (1981) suggested that the microenvironmental regulation of microtubule length might occur by a capping of their free ends near the cell surface. Anyway, the changes in microtubule length are evoked not only by changes in cell size, but also by some morphogenetic demands, and may be executed selectively in one category of microtubules only. This indicates that the control of microtubule length could be exerted through an appropriate MTOC, although along a different pathway than the control of microtubule number. It remains, however, quite uncertain whether the regulation of microtubule length is controlled by highly specific responses of different MTOCs to developmental signals, or through the formation of a highly elaborate pattern of microenvironments. This investigation was supported by the Polish Academy of Science, research grant no. II MR, PAN. I thank Dr Maria Jerka-Dziadosz for critical reading of the manuscript and Mrs Lidia Wiernicka for expert technical assistance. REFERENCES BAKOWSKA, J. (1980). Size dependent regulation of serially repeated structures of a protozoan Paraurostyla weissei. Ada Protozool. 19, BAKOWSKA, J. (1981). The ultrastructural analysis of the regulation of frontal cirri in Paraurostyla weissei. Ada Protozool. 20, BAKOWSKA, J., FRANKEL, J. & NELSEN, E. M. (1982). Regulation of the pattern of basal bodies within the oral apparatus of Tetrahymena thermophila. J. Embryol. exp. Morph. 69, BAKOWSKA, J. & JERKA-DZIADOSZ, M. (1980). Ultrastructural aspect of size dependent regulation of surface pattern of complex ciliary organelle in a protozoan ciliate.,7. Embryol. exp. Morph. 59, BRINKLEY, B. R., COX, S. M., PEPPER, D. A., WIBLE, L., BRENNER, S. L. & PARDUE, R. L. (1981). Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells, jf. Cell Biol. 90, DIDIER, P. (1970). Sur l'ultrastructureet lesmodalitfisd'elaboration desnfimadesmeschezquelques Cili6s Hym6nostomes Pfiniculiens Frontoniidae. Protistologica 6, GOLINSKA, K. (1978). The course of in situ remodelling of injured mouthparts mdileptus (Ciliata, Gymnostomata). Ada Protozool. 17,

15 Diminution oforganelles in small cells 39 GOLINSKA, K. (1979). Assessment of cell proportions during regeneration of Dileptus anser (Ciliata). Wilhelm Roux Arch. devlbiol. 187, GOLINSKA, K. (1982). Regulation of ciliary pattern in Dileptus (Ciliata). I. Sensory cilia and their conversion into locomotor cilia. J. Embryol. exp. Morph. 68, GOLINSKA, K. & BOHATIER, J. (1975). Action of Actinomycin D upon regenerative and divisional stomatogenesis in Dileptus. Ada Protozool. 14, GOLINSKA, K. & JERKA-DZIADOSZ, M. (1973). The relationship between cell size and capacity for division in Dileptus anser and Urostyla cristata. Ada Protozool. 12, GOLINSKA, K. & KINK, J. (1976). The regrowth of oral structures in Dileptus cygnus after partial excision. Ada Protozool. 15, GOLINSKA, K. & KINK, J. (1977). Proportional regulation of body form and cortical organelle pattern in the ciliate Dileptus. J. Cell Sci. 24, GRAIN, J. (1969). Le cinetosome et ses derives chez les Cilies. Ann. Biol. 8, GRAIN, J. & GOLINSKA, K. (1969). Structure et ultrastructure de Dileptus cygnus Claparede et Lachmann, 1859, Cili6 Holotriche Gymnostome. Protistologica 5, HAMMERSMITH, R. L. (1976). Differential cortical degradation in the two members of early conjugant pairs of Oxytrichafallax.J. exp. Zool. 196, JERKA-DZIADOSZ, M. (1976). The proportional regulation of cortical structures in a hypotrich ciliate Paraurostyla weissei.jf. exp. Zool. 195, JERKA-DZIADOSZ, M. (1977). Temporal coordination and spatial autonomy in regulation of ciliary pattern in double forms of a hypotrich ciliate Paraurostyla weissei.jf. exp. Zool. 200, JERKA-DZIADOSZ, M. & GOLINSKA, K. (1977). Regulation of ciliary pattern in ciliates.j. Protozool. 16, KINK, J. (1976). A localized region of proliferation in growing cells of Dileptus visscheri (Ciliata, Gymnostomata). jf. Cell Sci. 20, PAULIN, J. J. & BUSSEY, J. (1971). Oral regeneration in the ciliate Stentor coeruleus: A scanning and transmission electron optical study, jf. Protozool. 18, PEARSON, P. J. &TUCKER, J. B. (1977). Control of shape and pattern during the assembly of a large microtubule bundle. Evidence for a microtubule-nucleating-template. Cell Tiss. Res. 180, PICKETT-HEAPS, J. D. (1969). The evolution of the mitotic apparatus: an attempt at comparative ultrastructural cytology in dividing plant cells. Cytobios 3, RAFF, E. C. (1979). The control of microtubule assembly in vivo. Int. Rev. Cytol. 59, RUFFOLO, J. J. & FRANKEL, J. (1972). Observations on stomatogenesis in Tetrahymenapyriformis GL-C using scanning electron microscopy. J. Protozool. 19 (abstr.), 21. SAWYER, H. R. JR & JENKINS, R. A. (1977). Stomatogenic events accompanying binary fission in Blepharisma. jf. Protozool. 24, TUCKER, J. B. (1970). Morphogenesis of a large microtubular organelle and its association with basal bodies in the ciliate Nassula. jf. Cell Sci. 6, TUCKER, J. B. (1979). Spatial organisation of microtubules. In Microtubules (ed. K. Roberts & J. S. Hyams), pp New York, London: Academic Press. TUFFRAU, M. (1967). Perfectionnements et pratique de la technique d'imprdgnation au Protargol des Infusoires Cilies. Protistologica 3, (Received 23 August 1983 Accepted, in revised form, 9 March 1984)

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