Temperature-induced modifications in size and pattern of microtubular
organelles in a ciliate, Dileptus
I. Supernumerary microtubules in axonemes of sensory cilia
KRYSTYNA GOLINSKA
Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw 02-093, Poland
Summary
Supernumerary microtubules were found in the
so-called sensory cilia, in addition to a sensory
axoneme. The supernumerary microtubules were
not structurally connected to a basal body, but
were probably anchored to clusters of dense
material inside the ciliary shaft. The frequency
of appearance of the supernumerary microtubules was found to be temperature-dependent:
the higher the temperature during formation of
sensory cilia, the greater was the number of
supernumerary microtubules in cross-sections,
and the more cross-sections contained them.
The possibility is discussed that the formation
of the supernumerary microtubules is not due to
formation of new nucleating sites inside the
cilium. Instead, the microtubules may be remnants of a previously existing axoneme, separated from the basal body during the formation of
a sensory cilium. Some of the microtubules of the
released axoneme may persist as the supernumerary microtubules, if capped with dense
material or some other structure within the ciliary shaft.
Introduction
microtubules than the locomotor cilium. The supernumerary microtubules, if present, are not structurally linked to the basal body, but are anchored to
clusters of dense material (Phillips, 1979; Erler,
1983).
In this study supernumerary microtubules were
found in axonemes of the so-called sensory cilia in
Dileptus. These microtubules are not connected to the
basal body, but to some dense material within the
ciliary shaft. The frequency of appearance of these
microtubules, and their number per cross-section,
were found to increase with increasing temperature.
Data were analysed to determine the possible ways of
formation of the supernumerary microtubules.
Microtubule pattern in locomotor cilia is highly
conservative, but may be altered by expression of
some genie mutations (Forest, 1983), by the action of
taxol (Herth, 1983) or by cold treatment (Szollosi,
1976). Usually some microtubules are missing from
the modified axoneme. When supernumerary microtubules are present (Herth, 1983) the ciliary axoneme
is complete, and the site of attachment of these
additional microtubules must be some place other than
the basal body.
Cilia that have no locomotor function, such as the
primary cilia in embryonic tissue (Dalen, 1981),
sensory cilia of many receptors (reviewed by Barber,
1974; Altner & Prillinger, 1980), or those called the
sensory or clavate cilia in ciliates (Grain & Golinska,
1969; Holt eta/. 1973; Golinska, 1982, 1983), usually
have a modified microtubule pattern. When the pattern is changed, the basal body usually bears fewer
Journal of Cell Science 87, 349-356 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Key words: ciliates, supernumerary micr'otubules, cilia,
temperature.
Materials and methods
The material used in this study was Dileptus margaritifer, in
previous publications referred to as Dileptus anser (see
revision by Wirnsberger et al. 1984). Stock cultures were
kept at room temperature and fed every other day with
349
Colpidium spp. Details of culture methods have been
published (Golinska & Jerka-Dziadosz, 1973).
In this study posterior fragments (opimers) of Dileptus
cells were used. Transections were made in the middle of the
trunk, then the posterior fragments were isolated into
depression slides and placed in a thermostat adjusted to
10°C, or 32°C. Control fragments were left at room temperature. At 24 h after the operation all cells were fixed and
prepared for electron microscopy. Since the sensory cilia are
situated in the anterior region of the cell, all the sensory cilia
observed were those formed by opimers exposed to cold,
heat, or room temperature during the 24 h following the
operation. Data given for 'normal' cells were obtained from
micrographs made during previous studies on untreated cells
taken from growing cultures. Fig. 12 is part of a published
micrograph (see Golinska, 1983, fig. 22).
Fixation for electron microscopy was performed by mixing equal amounts of OsO* (4 %), glutaraldehyde (6 %), and
0-05 M-cacodylate buffer, pH 6-8. The mixture was prepared
immediately before fixation. During dehydration, 30%
ethanol containing 0-1 % tannic acid was applied for 30 min.
Further processing of the samples was standard. Preparations were examined in a JEM 100 B transmission electron microscope.
Sensory
cilia
Proboscis
. Cytostome
Locomotory "•
cilia
Trunk
Results
The cell of Dileptus consists of two main parts: the
trunk and its anteriorly located slender elongation, the
so-called proboscis. A cytostome is situated at the base
of the proboscis. The whole body except for the oral
region is covered with longitudinal rows of somatic
cilia. Several dorsally located rows contain locomotory
cilia in their posterior portion, while in their anterior
portion the pairs of sensory cilia can be seen situated
on the proboscis (Fig. 1). The fine structure of
these cilia, as well as the pathways of their formation
and transformation, have been described (Grain &
Golinska, 1969; Golinska, 1982, 1983). The cilia are
termed 'sensory' not because of their function, which
is unknown, but because of their structural simplification, which resembles that of cilia found in sensory
cells in chemo- and mechanoreceptors of higher organisms (reviewed by Gaffal & Bassemir, 1974; Altner &
Prillinger, 1980). The sensory cilia in Dileptus are
shorter than locomotor ones (Fig. 1), have no B
tubules (i.e. the 9 outer microtubules are single)
(Figs 4, 6, 7), and contain no intertubular links. This
is confirmed by the irregular distribution of microtubules within the sensory shafts (Figs 4, 6, 7, 10).
The sensory cilia of each pair are very much alike,
differing only in that the basal body of the posterior
cilium is equipped with additional root fibres (Fig. 2).
Most cross-sections of sensory cilia contain 11
microtubules, i.e. the two microtubules of the central
pair and nine outer single microtubules (Fig. 4). The
microtubules of the central pair are probably shorter
than the outer ones, since there are many sections
350
K. Golinska
Fig. 1. Side-view of Dileptus. One dorsal ciliary row is
shown, bearing sensory cilia in its anterior part, and
locomotor cilia in the posterior part.
containing nine and fewer microtubules. Such sections
were not taken into account. Sections showing 11
single microtubules and no central pair were frequently encountered. These were regarded as probably resulting from separation of the central pair and
classified as cilia without additional microtubules,
although we cannot exclude the possibility that such
sections represent axonemes with two additional
microtubules sectioned above the central pair. In
counting microtubules no discrimination was made
between outer microtubules and microtubules belonging to the central pair.
The additional microtubules were first observed in
sensory cilia of cells that regenerated their probosces
while exposed to elevated temperature. This led to
observations being made on sensory cilia formed by
opimers exposed to 32°C, 20°C and 10°C. Since my
equipment was rather primitive, possible temperature
deviations were ±2 deg. C. The results of microtubule
counting on cross-sections of sensory shafts are summarized in Table 1. The number of cells to which the
cilia belonged was always more than 20. The results
show clearly that elevation of temperature is followed
by an increase in the number of supernumerary
microtubules, while cold treatment causes reduction
in their number. At room temperature the additional
microtubules are formed in intermediate quantities,
similar to that found in normal cells.
Fig. 2. Normal cell. Section through pairs of sensory cilia belonging to three rows (marked by broken lines), a, Anterior;
p, posterior cilium in a pair. Arrows indicate root fibres connected only to the posterior basal body. X36 500.
Fig. 3. Longitudinal section of sensory cilium in normal cell, m, Additional microtubule. X50 000.
Fig. 4. Normal cell. Row of sensory cilia without additional microtubules. a, Anterior cilium; p, posterior cilium. X55 500.
Fig. 5. Normal cell. Row of sensory cilia containing additional microtubules. Microtubules are shown by short lines.
Numbers represent the total number of microtubules in the shaft. Additional microtubules (numbers higher than 11) can
be found in every other shaft in this row. X38 000.
Fig. 6. Cell 24h after the operation, 32°C. Sensory shaft with two additional microtubules (11+2). d, Dense substance in
contact with microtubule and ciliary membrane. X96 000.
Fig. 7. Cell 24h after the operation, 32°C. Sensory shaft with one additional microtubule (10+2). d, Dense material,
situated as in Fig. 6. X 108 000.
Supernumerary microtubules in sensory cilia
351
Table 1. Micmtubule content in sensory shafts
formed at different temperatures by posterior
fragments (opimers) and normal cells o/Dileptus
Group of cells
Opimers
32°C
n = 81
Pattern of
microtubules
10+2
Opimers
20°C
n = 91
Normal
cells
n = U9
Opimers
10°C
n = S4
o of cross-sections examined
9-9
4-4
6-7
3-3
5-5
12 to 14+2
14-8
8-6
7-4
Total
30-8
14-3
10-0
5-5
11+2
n = number of cross-sections.
The additional microtubules were never observed at
the level of the axial granule, and no modifications of
microtubule pattern were found in the basal bodies of
sensory cilia. It was always in the portion of a sensory
cilium above the axial granule that the supernumerary
microtubules were found. Within shafts containing
supernumerary microtubules some of the microtubules were usually in contact with masses of dense
material, often situated beneath the ciliary membrane
(Figs 6, 7). Similar clusters of dense material, but
without apparent connection with microtubules or
ciliary membrane, were often observed in sensory
shafts (Figs 8, 10), and also in shafts of locomotor cilia
(Figs 9, 11), where additional microtubules were
never encountered. No counts of the cilia containing
such dense material were made, but they can be found
in cells exposed to both elevated and low temperatures. The dense material may serve as the site of
attachment for additional microtubules in sensory
cilia.
The supernumerary microtubules, when present in
several cilia in one section, were always found in every
other cilium in the row, i.e. in one cilium of each
sensory pair (Fig. 5). In four cases grazing sections
were found, showing not only cilia with supernumerary microtubules, but also the proximal parts of basal
bodies in another sensory pair of the same region. This
enabled a distinction to be made between the anterior
and posterior cilium in a pair. In three cases the
additional microtubules were situated in the anterior
shaft, and in one case in the posterior sensory shaft.
This peculiar location of additional microtubules, in
only one cilium of the sensory pair, indicates that the
formation of these microtubules takes place during the
formation of the sensory cilium. As was reported
earlier (Golinska, 1983), the sensory unit is formed
by transformation of a locomotor unit: through the
resorption of a locomotor cilium (leaving its basal body
352
A'. Golinska
intact), formation of a basal body for the anterior
cilium, and formation of sensory shafts for both basal
bodies of the pair. The additional microtubules, when
situated in the posterior ciliary shaft, may represent
the remnants of an old locomotor cilium, not properly
resorbed.
A possible explanation of the presence of additional
microtubules in the anterior ciliary shaft came from a
confirmation of the earlier observation (Golinska,
1983) that there is no definite sequence of anterior
basal body formation, resorption of the locomotor
cilium and formation of sensory cilia. In the region
where the sensory units are forming, basal body
formation may precede or follow resorption of the
locomotor cilium, and anterior sensory cilia may be
found with posterior cilia not yet resorbed, with
posterior cilia undergoing resorption, or with posterior
newly formed sensory cilia (Figs 12, 13). It was in the
first configuration that a longitudinal section of a
developing sensory pair was found, showing a separation of the new sensory axoneme from the anterior
basal body (Fig. 14), possibly in response to the
resorption process beginning in the locomotor axoneme of the posterior cilium. A probable later stage
is presented in Fig. 15, where sensory shafts have
already formed on both basal bodies, and supernumerary microtubules in the anterior one are distally
displaced.
The complete lack of additional microtubules in
locomotor cilia, in spite of the presence of dense
material in their shafts (Figs 9, 11), is further confirmation that the supernumerary microtubules are not
nucleated at clusters of dense material (or some other
structure, i.e. ciliary membrane), but are the remnants of previously existing axonemes.
Discussion
Several questions concerning the supernumerary
microtubules seem to be of interest, namely their sites
of nucleation, their temperature sensitivity, and their
bearing upon the morphogenesis of microtubular
organelles.
The site of nucleation of supernumerary microtubules may be represented either by the basal body,
or some other structure where the microtubules are
anchored, i.e. dense material or ciliary membrane.
The supernumerary microtubules in sensory cilia of
Dileptus are, in my opinion, nucleated at basal bodies,
being the remnants of an axoneme separated from the
basal body and later replaced by a set of microtubules
forming a new axoneme (Fig. 16). The presence of
additional microtubules in only one cilium of the pair
indicates that their formation takes place during the
formation of a sensory unit. The separation of the
locomotor axoneme from the posterior basal body
Fig. 8. Longitudinal section of sensory shaft in normal cell, d, Dense material. X41 000.
Fig. 9. Longitudinal section of locomotor cilium in normal cell, d, Dense substance. X 68 500.
Fig. 10. Cross-section of sensory cilium formed by the cell at 32°C, 3h after the operation, d, Dense material, x 110 500.
Fig. 11. Cross-section of locomotor cilium in a cell treated with 32°C during 24 h. d, Dense material. X96000.
Fig. 12. Grazing section of forming sensory cilia 50— 80min after the operation, room temperature, a, Anterior cilium;
p, posterior cilium. The upper left pair contains both cilia of sensory type; the lower right pair has, instead of a posterior
cilium, a bulbous mass with numerous vesicles, representing the resorption of old locomotory cilium. X52 000.
Fig. 13. Sensory pair in regenerating cell 30—60min after the operation, room temperature, a, Anterior sensory shaft;
p, posterior shaft contains locomotory-type axoneme. X58 500.
Supernumerary microtubules in sensory alia
353
Fig. 14. Forming sensory pair in regenerating cell
90-120 min after the operation, room temperature. In the
anterior cilium (a) a new sensory axoneme is separated
from the basal body; p, posterior cilium with locomotor
axoneme. X 57 500.
is a normal event in ciliogenesis of sensory cilia
(Golinska, 1983). Separation of the sensory axoneme
from the anterior basal body may be induced when the
resorption of a posterior locomotor cilium coincides
with the presence of an already developed anterior
sensory cilium (Fig. 16). Since the formation of additional microtubules is rather uncommon (in the
posterior cilium as well as in the anterior one), their
occurrence in both components of a sensory pair may
be extremely rare, giving the impression that additional microtubules can be found only in one cilium
of a pair. This pattern of occurrence of additional
microtubules would be inexplicable if they were nucleated at some structure other than a basal body.
This speculation concerning the sites of nucleation
of supernumerary microtubules is further supported
by several observations. The total number of microtubules per sensory shaft was always less than two sets
of microtubules; in fact, the highest number of
additional microtubules was five. This, again, may be
easily explained only when the basal body is accepted
as the site of nucleation of all microtubules in the
ciliary shaft, nucleation at any other structure being
theoretically able to generate an unlimited number of
microtubules. Also a complete lack of supernumerary
microtubules in locomotor cilia (well equipped with
structures representing possible nucleating sites)
speaks in favour of the basal body as the site of
nucleation of additional microtubules.
354
K. Golinska
Fig. IS. Growing sensory cilium in regenerating cell
120—150 min after the operation, room temperature.
Arrows indicate structures that may represent additional
microtubules. x59 000.
There is a possibility that the formation of additional microtubules by separation of the axoneme
from the basal body, may not be found in Dileptus
only. The observations of Herth (1983) on taxolinduced formation of supernumerary microtubules in
flagella has revealed that besides single microtubules,
supernumerary doublets and central pairs were also
formed. It seems very unlikely that such structures
would be nucleated at some structure other than a
basal body. In the same study the taxol-induced
resorption of flagella was reported as being frequent
(Herth, 1983).
In this study temperature was found to influence
both the number and the frequency of appearance of
additional microtubules. This means that either there
is an alteration in the number of cilia containing the
supernumerary microtubules, or a change in length of
these microtubules, or both. Whichever is the case, the
additional microtubules show higher sensitivity to
temperature than the microtubules of the sensory
shaft. This may be directly related to the stability of
microtubules, which is well known from in vitro
studies to be temperature-sensitive (review by Scheele
& Borisy, 1979). In vivo the microtubules show
differences in their sensitivity to temperature, even if
I
Fig. 16. Proposed ways of formation of supernumerary microtubules (arrows) in the posterior sensory cilium (upper row)
and in the anterior sensory cilium (lower row).
anchored at the same microtubule-organizing centre
(MTOC) (Jones & Tucker, 1981; Schultze & Kirschner, 1986). Different sensitivity for microtubules
anchored at different structures, like those of the
sensory shaft and supernumerary microtubules in
Dileptus, seems highly likely. Another possibility is
that the temperature sensitivity of supernumerary
microtubules is a consequence of a temperature effect
upon the process of ciliary resorption (which is known
to be temperature-dependent: see Hinrichson, 1981),
or upon the process of capping the microtubules with
dense material or some other structure.
The idea that microtubules can be nucleated at one
site, and afterwards anchored at another site, has some
interesting consequences. For microtubular organelles
in ciliates it is generally accepted that a MTOC is both
nucleating and capping the microtubules (Tucker,
1982). A second function for a MTOC, that of
anchoring and thereby stabilizing the microtubules
nucleated elsewhere, was proposed by Kirschner
(1980). Basal bodies in sensory cilia of Dileptus
operate as a MTOC of the first kind upon microtubules of the sensory axoneme, while the function of
the MTOC of the second kind is fulfilled by some
other structure, i.e. by clusters of dense material in the
sensory axoneme. The basal bodies of sensory cilia, in
their formation of consecutive generations of microtubules, resemble the dynamic model of the centrosome presented by Schultze & Kirschner (1986), but
differ from the model in the formation of separate
populations of microtubules from these microtubule
generations.
This work was supported by grant no. C.P.B.P. 04.01
from the Polish Academy of Sciences. I thank Dr A. V.
Grimstone and Dr Maria Jerka-Dziadosz for critical reading
of the manuscript, and Mrs Lidia Wiernicka for expert
technical assistance.
References
ALTNER, H. & PRILUNGER, L. (1980). Infrastructure of
invertebrate chemo-, thermo-, and hygroreceptors and its
functional significance. Int. Rev. Cytol. 67, 69-139.
BARBER, V. C. (1974). Cilia in sense organs. In Cilia and
Flagella (ed. M. A. Sleigh), pp. 403-433. New York,
London: Academic Press.
DALEN, H. (1981). An ultrastructural study of primary
cilia, abnormal cilia and ciliary knobs from the ciliated
cells of the guinea pig trachea. Cell Tiss. Res. 220,
685-697.
Supernumerary microtubules in sensory cilia
355
ERLER, G. (1983). Sensitivity of an insect mechanoreceptor
after destruction of dendritic microtubules by means of
vinblastine. Cell Tiss. Res. 229, 673-684.
FOREST, C. L. (1983). Mutational disruption of the 9+2
structure of the axoneme of Chlamydomonas flagella. J.
Cell Set. 61, 423-436.
GAFFAL, K. P. & BASSEMIR, U. (1974). Vergleichende
Untersuchung modifizierter Cilienstrukturen in den
Dendriten mechano- und chemosensitiver Rezeptorzellen
der Baumwollwanze Dysdercus und der Libelle Agrion.
Protoplasma 82, 177-202.
GOLINSKA, K. (1982). Regulation of ciliary pattern in
Dileptus (Ciliata). I. Sensory cilia and their conversion
into locomotor cilia. J . Embryol. exp. Morph. 68,
99-114.
GOLINSKA, K. (1983). Regulation of ciliary pattern in
Dileptus (Ciliata). II. Formation of a cortical domain of
sensory cilia from a domain of locomotor cilia, jf. Cell
Sci. 62, 459-475.
GOLINSKA, K. & JERKA-DZIADOSZ, M. (1973). The
relationship between cell size and capacity for division in
Dileptus anser and Urostvla cristata. Ada protozool. 12,
1-21.
GRAIN, J. & GOLINSKA, K. (1969). Structure et
infrastructure de Dileptus cygnus Claparede et
Lachmann, 1859, Cili6 Holotriche Gymnostome.
Protistologica 5, 269-291.
HERTH, W. (1983). Taxol affects cytoskeletal microtubules,
flagella and spindle structure of the chrysoflagellate alga
Poterioochmmonas. Protoplasma 115, 228-239.
HINRICHSEN, R. D. (1981). An analysis of specific
temperature blocks in the conjugation sequence of
Paramecium tetraurelia. J. Protozool. 28, 417-423.
HOLT, P. A., LYNN, D . H. & CORUSS, J. O. (1973). An
ultrastructural study of the tentacle-bearing ciliate
356
K. Golinska
Actinobolina smalli n.sp. and its systematic and
phylogenetic implications. Protistologica 9, 521-541.
JONES, J. C. R. & TUCKER, J. B. (1981). Microtubule-
organizing centres and assembly of the double-spiral
microtubule pattern in certain heliozoan axonemes. J.
Cell Sci. 50, 259-280.
KIRSCHNER, M. W. (1980). Implications of treadmilling for
the stability and polarity of actin and tubulin polymers in
vivo. J. Cell Biol. 86, 330-334.
PHILLIPS, D. W. (1979). Infrastructure of sensory cells on
the mantle tentacles of the gastropod Notoacmea scutum.
Tissue & Cell 11, 623-632.
SCHEELE, R. B. & BORISY, G. G. (1979). In vitro assembly
of microtubules. In Microtubules (ed. K. Roberts & J.
S. Hyams), pp. 175-254. New York, London: Academic
Press.
SCHULTZE, E. & KIRSCHNER, M. W. (1986). Microtubule
dynamics in interphase cells. J. Cell Biol. 102,
1020-1031.
SZOLLOSI, A. (1976). Influence of infra-optimal breeding
temperature on spermiogenesis of the locust Locusta
migratoria. I. Abnormalities in differentiation of the
cytoplasmic organelles. J. Ultrastruct. Res. 54, 202-214.
TUCKER, J. B. (1982). Microtubule-organizing centres and
assembly of intricate microtubule arrays in protozoans.
In Microtubules in Microorganisms (ed. P. Cappuccinelli
& N. R. Norris), pp. 15-29. New York: Marcel Dekker
Inc.
WlRNSBERGER, E., FOISSNER, W. & ADAM, H . (1984).
Morphologie und Infraciliatur von Perispira pyriformis
nov. spec, Cranotheridiumfoliosus (Foissner, 1983) nov.
comb, und Dileptus anser (O. F. Muller, 1786)
(Protozoa, Ciliophora). Arch. Protistenk. 128, 305-317.
(Received 11 September 1986 -Accepted 24 November
1986)