J. Cell Set. ai, 35-46 (1976)
Printed in Great Britain
35
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 characteristic 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 reestablishing 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. Kaczanowska (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 W T , clone TC-2, was cultured axenically in 2 % (w/v) proteosepeptone 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 proceeded. 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 interferencecontrast.
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 considerably 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 fibrelattice. 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 preexisting 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,
I0
74)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 proceeds (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
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 sequence 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 Fellowship awarded by the National Research Council of Canada.
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