J. Cell Sci. 44, 135-151 (1980)
Printed in Great Britain © Company of Biologists Limited 1980
135
MICROTUBULES AND CONTROL OF
MACRONUCLEAR 'AMITOSIS' IN PARAMECIUM
J. B. TUCKER,* J. BEISSON.f D. L. J. ROCHE*
'Department of Zoology, The University, St Andrews,
Fife KY16 gTS, Scotland
AND J. COHENf
f Centre de Ginitique Moliculaire, Centre National de la Recherche Scientifique,
91190 Gif-sur-Yvette, France
SUMMARY
The ' amitotic' division of the macronucleus during binary fission in P. tetraurelia includes
a detailed sequence of shape changes that are temporally coordinated with the adoption of a
series of well-defined positions and orientations inside the cell. The deployment of nucleoplasmic microtubules that is spatially correlated with the shaping ritual is more complex and
precise than has been reported previously. Macronuclear division is not amitotic. It is not a
simple constriction into two halves.
As a dividing macronucleus starts to elongate it becomes dorsoventrally flattened against the
dorsal cortex of the organism and assumes an elliptical shape. Concurrently, an elliptical marginal band of intranuclear microtubules assembles that has the same spatial relationship to nuclear
shape as the marginal microtubule bands of certain elliptical vertebrate blood cells have to cell
shape. The band breaks down as further elongation occurs and the nucleus adopts the shape of
a straight and slender sausage. Most of the intranuclear microtubules assemble as elongation,
starts and break down shortly after elongation is completed; the majority are oriented parallel
to the longitudinal axis of the nucleus throughout elongation. Some of them are attached to
nucleoli and are coated with granules which are almost certainly derived from the cortices of
nucleoli. The peripheral concentration, interconnexion, orientation, and overlapping arrangement of microtubules, and the reduction in microtubule number per nuclear cross-section
as elongation proceeds at a rate of about 40 /ttn min"1, are all compatible with the provision
of a microtubule sliding mechanism as the main skeletal basis for elongation. There are indications that this mechanism is augmented by anchorage and/or active propulsion of nucleoli
that may perhaps facilitate fairly equitable segregation of chromosomal material to daughter
nuclei.
INTRODUCTION
Division of the highly polyploid varieties of ciliate macronuclei is universally
referred to as an amitotic procedure. An amitotic division is one in which a nucleus
divides by simple constriction into 2 halves without formation of a spindle or dissolution of the nuclear envelope (Abercrombie, Hickman & Johnson, 1973) and without
regular segregation of chromosomal material (Raikov, 1969). The nuclear envelope
does remain intact during division of macronuclei, and although these elongating
nuclei contain, or are surrounded by, large numbers of microtubules oriented parallel
to their longitudinal axes the tubules do not form conventionally constructed mitotic
spindles (Carasso & Favard, 1965; Tucker, 1967; Jurand & Selman, 1969, 1970;
136
J. B. Tucker, J. Beisson, D. L. J. Roche and J. Cohen
Millechia & Rudzinska, 1971; Stevenson & Lloyd, 1971; Inaba & Kudo, 1972;
Walker & Goode, 1976; Jenkins, 1977). However, Raikov (1969) and Grell (1973)
have pointed out that it may be incorrect to describe macronuclear divisions as
amitotic because of the possibility that genetic information is distributed more
precisely during these divisions than was originally supposed. This report supports
their views in so far as it establishes that macronuclear division in Paramecium
tetraurelia is not just a simple constriction into 2 halves. A detailed and highly coordinated programme of intranuclear microtubule deployment, and nuclear shaping
and positioning, is involved.
Analysis of macronuclear shaping and positioning is pertinent to the study of 2
widespread but incompletely understood phenomena in cells generally. One is the
cytoskeletal basis for precise nuclear positioning and orientation in the cytoplasm of
certain cells (for example, Meats & Tucker, 1976). The other, albeit less obvious, is
the role of microtubules during control of cell shaping (for review see Tucker, 1979),
because this investigation reveals that some aspects of the spatial involvement of
microtubules in the shaping of a protozoan nucleus show very close correspondence to
those that occur during certain types of metazoan cell shaping. P. tetraurelia is
especially favourable material for analysis because of the availability of non-lethal
mutants that interfere with these and related phenomena during macronuclear division
(Sonneborn, 1974; Beisson & Rossignol, 1975; Ruiz, Adoutte, Rossignol & Beisson,
1976). Such mutants have not been obtained for other cells. This paper establishes a
basis for such analysis; it gives a detailed account of the normal course of events during
macronuclear division in wild-type P. tetraurelia. It provides evidence for the role
of microtubules in shape control that is supported by an accompanying report (Cohen,
Beisson & Tucker, 1980) on abnormal microtubule deployment during defective
macronuclear division in the tarn 8 mutant of the same organism.
MATERIALS AND METHODS
Culture
Paramecium tetraurelia stock dd, — 2 (Sonneborn, 1974, 1975) is a derivative of stock 5/
carrying the allele k in the stock J J genetic background. Paramecia were cultured in Scotch
Grass infusion which was inoculated with Klebsiella pneumoniae 24 h before inoculation with
Paramecium.
Electron microscopy
Paramecia were prepared for electron microscopy using procedures already described
(Tucker, 1967). Organisms at early stages (1-2, see Fig. 1) of binary fission do not have a
clearly detectable cleavage furrow and are not readily distinguishable frominterfission organisms when examined using light microscopy after fixation and flat embedding in resin for electron
microscopy. Living organisms at stages 1 and 2 possess a slight bulging of the cell body near
the mid-region that is detectable when such organisms are examined with a stereo-binocular
dissecting microscope. These organisms were isolated individually by pipetting from cultures
into fixative and then individually prepared for electron microscopy. Later fission stages were
selected on the basis of the stage of development of cleavage furrows (which are well correlated
temporally with the progress of nuclear division; see Fig. 1) from cultures fixed during logphase growth that included all stages in the asexual binary fission cycle.
Macronuclear microtubules
137
The number and distribution of microtubules in thin cross-sections of dividing macronuclei has been assessed by marking their positions on electron micrographs at final print
magnifications of x 30000. Prints were prepared from negatives taken at microscope magnifications of x 15000. Several such negatives and their prints were required to produce a
complete cross-sectional montage from negatives at this magnification (which is the minimum,
that routinely permitted the sufficiently accurate focusing of the microscope needed to resolve
microtubules clearly). Accurate juxtaposition of prints and elimination of 'overlap' during
preparation of each montage was achieved by using nucleoli (which can be distinguished from
each other on the basis of the different shapes and sizes of their profiles in the sections) as
indicators of the margins of regions only included in one negative contributing to a montage.
The cross-sections employed were all cut at points along nuclei where their cross-sectional
areas were maximal, near the mid-region of each nucleus at stages 2-4, and the mid-regions of
putative daughter nuclei (avoiding the narrow central isthmus) at stage 6 (Fig. 1).
Light microscopy
Additional assessments of changes in the shapes, dimensions, and intracellular positions of
macronuclei during the fission cycle, and the temporal correlation of these changes with the
other main structural events associated with fission (Fig. 1), are based on light-microscopical
examination of fixed organisms prepared using Azure-A, and Dippell's staining procedures
(see Cohen et al. 1980). The time elapsing between the start of fission (stage 1) and each succeeding division stage was assessed by examination of individually isolated living paramecia.
The progress of changes in cell shape and length was followed for 18 organisms isolated at
stage 1 or earlier from log-phase cultures maintained at 27 °C. In some of these organisms
changes in the lengths of macronuclei could also be observed.
RESULTS
The main stages in the shaping and positioning of dividing macronuclei are summarized in Fig. 1. The macronuclei of interfission organisms and organisms at 4
stages of division (2, 3, 4 and 6) were examined. The investigation concentrated
mainly on stages 3 and 4 (Table 1) because these span the period of most marked
elongation and shape change.
The shape of an interfission macronucleus approximates to that of a prolate
spheroid. No microtubules were detected in interfission nuclei. As fission starts
(stages 1 and 2) a macronucleus 'condenses' slightly (Stevenson & Lloyd, 1971). It
loses its elongate spheroidal shape (and becomes more compact and spherical) and
begins to migrate dorsally and anteriorly from its characteristic interfission position
against the gullet. Cross-sections of a stage 2 macronucleus revealed that microtubules
had started to assemble in the nucleoplasm and appeared to be randomly oriented. The
presence of well-defined anisometric macronuclear organization is first apparent at
stage 3. The nucleus is elongate, oriented parallel to the organism's longitudinal
axis, and the number of microtubule profiles/nuclear cross-section (N) (Table 1) is
much greater (584-825) than it is at stage 2 (190). The percentage of longitudinally
oriented (i.e. cross-sectioned) microtubules/nuclear cross-section (as a percentage of
JV, see Table 1) lies in the range 72-82%. Serial sectioning revealed that stage 3
nuclei are dorsoventrally flattened, elliptical (about 60 /jm long and 25 /tm wide),
and closely applied to the dorsal cortex (Figs. 2, 7). The nucleus is slightly curved in
a dorsoventral direction about its longitudinal axis so that the dorsal surface of the
nuclear envelope is convex, more or less concentric with the curvature of the dorsal
10
CEL
44
J. B. Tucker, J. Beisson, D. L. J. Roche andj. Cohen
Postfission
Stage 6
18 min
Stage 5
15 min
Fig. i. Nuclear events during the cell cycle of Paramedum tetraurelia. Schematic
diagram showing changes in the length, shape and position of a macronucleus (ma)
during the cell cycle, including the 6 division stages referred to in the text, based on
light- and electron-microscopical examinations. The spatio-temporal relationship of
these changes to elongation and cleavage of the cell body, and the positioning of each
micronucleus (mi) and oral apparatus (oa) are also shown. The dorsal surfaces of organisms are oriented towards the left side of the diagram and the anterior poles towards the
top. The average times elapsing (based on in vivo observations, see Materials and
methods) from stage i until each succeding division stage are included. The positions
and orientations of the macronuclear cross-sections shown in Figs. 2, 3 and 8 are
indicated by short arrows.
Stage i. The macronucleus rounds up, micronuclei are at mitotic prophase, a slight
bulging of the cortex starts to appear in the anterior portion of the organism and the
oral apparatus is already duplicated (Kaneda & Hanson, 1974).
Stage 2. The macronucleus migrates towards the anterior portion of the dorsal cortex and the anterior bulging is more marked. The metaphase micronuclei are
apparently not confined to any particular cytoplasmic region.
Stage 3. The macronucleus becomes dorsoventrally flattened and elliptically shaped
(shown in side view here). It elongates from the anterior pole towards the posterior
of the organism. The post-telophase micronuclei start to elongate and the new oral
apparatus in the opisthe starts to migrate away from the old one. The cleavage furrow
is first detectable at this stage.
Stage 4. The sausage-shaped macronucleus continues to elongate.
Stage 5. The macronucleus starts to constrict in the cleavage furrow plane and
pairs of daughter micronuclei transiently take up polar locations.
Stage 6. The cleavage constriction is less than 5 /(in across, daughter macronuclei
have separated in some organisms and micronuclei lose their apical locations.
Postfission.After separation of daughter organisms the macronucleus soon adopts the
shape of an elongate spheriod and takes up a position against the oral apparatus again.
Macronuclear microtubules
'*
**r •$/4''V
Fig. 2. Cross-section through a dorsoventrally flattened stage 3 macronucleus that is
closely positioned against the dorsal cortex. The dorsal surface of the nuclear envelope
is slightly indented where it contacts the proximal ends of trichocysts (arrows), x 7000.
140
J. B. Tucker, J. Beisson, D. L. J. Roche and J. Cohen
Macronuclear truerotubules
I
141
10 jim
Fig. 7. Schematic diagram showing the shape and size of the macronucleus at stage 3
with a portion removed to show the cross-sectional profile of the elliptical nucleus at
its widest point and the lateral positions of the microtubules of the marginal band
with respect to this profile. The elliptical path followed by the band around the
periphery of the nucleus is also indicated. The line bearing arrowheads shows the
orientation of the organism's longitudinal (polar) axis. The other microtubules in the
nucleoplasm at this stage have not been included.
pellicle, and makes contact with the proximal ends of trichocysts attached to the
dorsal pellicle (Fig. 2). No cytoplasmic filaments or microtubules that might be
involved in producing or maintaining this dorsal positioning by connecting the
nucleus to components anchored in the dorsal cortex were detected. Elliptical stage
3 macronuclei contain an elliptically shaped marginal band of microtubules (Figs. 3,
6, 7). Cross-sections of nuclei reveal that each portion of a band includes about 70
microtubules that are more closely packed together than most of the microtubules
situated elsewhere in dividing nuclei. Some of them are interconnected by densely
staining material (Fig. 6).
Between stages 3 and 4 the macronucleus doubles in length (from about 60 /tm
up to 120 fim) and adopts the shape of a long and slender sausage (Fig. 1). This
phase of elongation is rapid. It was accomplished within 90 s in those living organisms
Fig. 3. Cross-section through part of a stage 3 macronucleus grazing through one of
its polar-directed extremities so that the marginal microtubule band is sectioned
longitudinally, x 58000.
Fig. 4. Cross-section of a bundle of peripheral microtubules in a stage 4 macronucleus.
Some of the tubules in such bundles are joined together by fine intertubule links
(arrow), x 260000.
Fig. 5. A granule-coated microtubule cut in cross-section in a stage 3 macronucleus.
x 292000.
Fig. 6. Cross-section through a portion of a stage 3 macronucleus at one of the lateral
extremities of its dor3oventrally flattened cross-sectional profile (compare Fig. 2),
showing the compact grouping of the marginal band microtubules. Some of these
tubules are joined together by dense material (arrows), x 125000.
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J. B. Tucker, J. Beisson, D. L. J. Roche and J. Cohen
*•
" 8
Fig. 8. Cross-section through a stage 4 macronucleus that is closely positioned against
the dorsal cortex shown at the same magnification as the stage 3 macronucleus in Fig.
2. The dorsal surface of the nuclear envelope forms several longitudinally oriented
pleats (arrows) that are cut in cross-section here, x 7000.
Fig. 9. A portion of the dorsally pleated nuclear envelope (<) of a cross-sectioned stage
4 macronucleus where it lies against the proximal ends of trichocysts (t). x 32000.
Macronuclear microtubules
143
Table 1. Quantitative data based on examination of cross-sections
of wild-type macronuclei
Microtubule
Cross-sectional
Internal
no./cross-section area of nucleus, microtubules,
Division stage of nucleus, N
/im1
%
2
190
140
38
3)
584
95
3/
534
825
72
127
25
26
565
3
4\
4/
4\
4i
4
1
4/
6
522
466
463
385
347
15
Longitudinally
oriented
microtubules,
%
49
74
72
17
82
48
45
54
58
48
36
93
84
94
96
27
27
21
15
93
82
64
20
96
31
25
Braces indicate that values were taken from sections of the same organism separated by
distances of at least 5 /im along the macronuclear longitudinal axis. Microtubules were considered to be longitudinally oriented if they exhibited well-defined circular cross-sectional
profiles in cross-sections of nuclei; tubules which exhibited longitudinal or oblique profiles are
therefore not included in this category but do contribute to the values recorded for N and
internal microtubules.
examined (see Materials and methods) in which the macronucleus was visible. By
stage 4 a nucleus is approximately circular in cross-section but is still closely applied
to the dorsal cortex (Fig. 8). Its cross-sectional area (Table 1) and shape vary to only
a small degree along most of its length, except at its ends where it rounds off fairly
abruptly (Fig. 1). Correlated with the loss of marked dorsoventral flattening and
elliptical shaping is the absence of a marginal microtubule band at this stage. Stage
4 nuclei have smaller cross-sectional areas (27-58 /im2) than stage 3 nuclei (72-127/6IT12)
and the range of values for N falls from 534-825 to 347-565 (Table 1). Most of the
microtubules (72-96%) in elongating stage 3 and 4 nuclei are oriented parallel to the
longitudinal axes of nuclei (Table 1). As in P. primaureUa (Stevenson & Lloyd, 1971),
most of the microtubules are situated close to the nuclear envelope. Those situated
within 1 /im of the nearest portion of the envelope will be referred to as peripheral
microtubules to distinguish them from the remainder which will be called internal
microtubules in the account that follows. There is no obvious abrupt change in microtubule arrangement or spacing at the 1 /im level. The distinction is a purely arbitrary
one that is useful for comparing tubule distribution at different stages. Use of this
criterion reveals that there is definitely a non-random distribution of microtubules
inside elongating macronuclei; most of the microtubules are concentrated within
1 /im of the envelope (Fig. 13). There are 17-26 % internal microtubules at stage 3 and
15-48% at stage 4 (Table 1). An examination of a nearly perfectly circular crosssectional profile of a stage 4 nucleus revealed that in this portion of the nucleus 40 %
144
J. B. Tucker, J. Beisson, D. L. J. Roche and J. Colien
&?* n
Macronuclear microtubules
145
Fig. 13. Schematic diagram of a portion of a stage 4 macronucleus showing the dorsal
pleating of its envelope and a typical distribution (black dots) for microtubule crosssectional profiles in relation to the cross-sectional profile of the nucleus. The lateral
profiles of peripheral tubules are depicted as if the envelope were transparent along
one side of the nucleus to show the meshwork of overlapping tubules revealed by
grazing surface sections of the nucleus.
by volume of the nucleoplasm (a peripheral i-/tm-thick shell just inside the envelope)
contained 75 % of the microtubules. No indications of the mechanisms responsible
for orienting tubules or concentrating them at the nuclear periphery were obtained.
Some of the peripheral microtubules in elongating nuclei are grouped together
to form small bundles while others are separated by distances of up to 0-5 /tm from
their nearest neighbours. In some of the bundles, tubules are joined together by
intertubule links (Fig. 4). Grazing longitudinal sections of elongating nuclei indicate
that peripheral tubules may form a structurally contiguous framework around the
nuclear periphery by virtue of intertubule linkage and their overlapping arrangement
(Fig. 10). These microtubules appear to form a meshwork of overlapping tubules in
which individual tubules splay away from bundles and then interdigitate between
the tubules of other bundles (Fig. 13). Some of the internal microtubules also form
bundles. These are more sparsely distributed than those at the periphery; whether
they also form part of an anastomosing meshwork has not been ascertained.
Some of the peripheral tubules are coated along considerable portions of their
Fig. 10. A portion of the periphery of a stage 4 macronucleus cut in longitudinal section. Peripheral microtubules have an overlapping arrangement and some of them are
coated with dense granules. One of the tubules (arrows) splays away from a microtubule bundle and converges with another bundle, x 63000.
Fig. i i . Part of a longitudinally sectioned stage 4 macronucleus showing a microtubule that is attached to a nucleolus (n) and coated with dense granules that are very
similar in appearance to those in the nucleolar cortex, x 96000.
Fig. 12. Part of a nucleolus with a microtubule running through its core in a cross-sectioned stage 3 macronucleus. x 417000.
146
J. B. Tucker, J. Beisson, D. L. J. Roche and J. Cohen
lengths with dense granules (Figs. 10, n , 14). The coating appears to be one or two
granules deep for most such portions (Fig. 5). These granules are similar in density
and dimensions to the maturing ribonucleoprotein pre-ribosomal particles that are
concentrated in the cortices of the numerous macronuclear nucleoli. Some of the
nucleoli were attached to peripheral microtubules in all of the stage 3 and 4 macronuclei examined (6 organisms). In most cases tubules were associated with only the
cortices of nucleoli (Figs. 11, 14) but in one instance a tubule passed through the core
of a nucleolus (Fig. 12). Examples of the latter type of association may not be uncommon. They would be difficult to detect except in cases like the one illustrated
where the microtubule passes through a lacuna in the dense core. Microtubuleattached nucleoli are usually situated close to granule-coated portions of tubules and
in some cases the coating is continuous with the granular nucleolar cortex (Figs. 11,
14). Sonneborn (1953) has estimated that mature interfission macronuclei in P.
tetraurelia contain between 500 and 2000 nucleoli. It is difficult to assess what proportion of the nucleoli are attached to microtubules at any one time, since thin
sections are about 2 microtubule diameters thick and nucleoli have diameters up to
25 times greater than this. Since convincing micrographs of tubule/nucleolus associations are provided only by sections that include a longitudinal profile of a tubule where
it is attached to a nucleolus, it is to be expected that such micrographs would be
obtained only with low frequency. Six microtubule/nucleolus attachments were
detected in a serial sequence of longitudinal sections cut through an entire stage 4
macronucleus with a diamond knife. Because of losses during section collection, and
those occurring as a result of support film instability and obscuration by grid bars,
approximately 25 % of this sequence was not examined.
Stage 4 nuclei possess longitudinally oriented slender tapering projections from their
lateral surfaces (one or two were found in all of the stage 4 nuclei examined). These
lateral projections each include a compact bundle of microtubules which runs from
the main body of the nucleus into the envelope-sheathed projection (Fig. 15). The
walls of adjacent tubules appear to make contact and/or to be interconnected by
dense intertubular material (Fig. 16). In this respect they resemble some of the
microtubules in the marginal band of stage 3 nuclei (Fig. 6) but differ from all the
other bundles in stage 4 nuclei (Fig. 4). Hence, lateral projection microtubule bundles
may represent remnants of the marginal band. Because of their interconnexion and
Fig. 14. Longitudinal section of a peripheral microtubule; its granular coating is
continuous with that of the attached nucleolus (ft). Stage 4. x 83 000.
Fig. 15. Longitudinal section through part of a stage 4 macronucleus grazing through
its lateral surface and cutting a micro tubule-containing lateral projection in longitudinal section, x 42000.
Fig. 16. Cross-section through a longitudinally oriented microtubule-containing
lateral projection from a stage 4 macronucleus. The closely packed microtubules are
sheathed by an evagination of the nuclear envelope and appear to be joined together by
dense material. This section formed part of a sequence which showed that the 5
microtubules in the projection extended into the main body of the nucleus 0-75 ft,m
from the level at which this section was cut. x 180000.
Macronuclear microtubules
147
•
'
:
*
148
J. B. Tucker, J. Beisson, D. L. J. Roche andj. Cohen
close packing they may be stiffer than the other microtubule bundles. Possibly they
are not fully integrated into the peripheral microtubule meshwork and are pushed
against the envelope during motile interactions associated with nuclear elongation to
stretch the envelope locally and form lateral projections.
There are other indications that the envelope is readily deformed during nuclear
elongation. The dorsal surface of the envelope is slightly indented at stage 3 where it
makes contact with the proximal ends of trichocysts (Fig. 2). Deformation is more
marked during stage 4 than stage 3 after considerable elongation of the dorsally
positioned nucleus has occurred; dorsal envelopes are extensively folded in most
cases (Figs. 8, 9). Sequences of transverse and longitudinal sections reveal that the
folds represent longitudinally oriented pleats in the envelope rather than a set of
digital evaginations (Fig. 13).
Elongation is nearly completed at stage 4. During stages 5 and 6 the macronucleus
thins out centrally prior to the final pinching off of daughter nuclei (Fig. 1). It
remains more or less circular in cross-section along its entire length during these
changes but increases in cross-sectional area on either side of the central isthmus in
regions that correspond to the mid-portions of the putative daughter nuclei. The
nucleus is somewhat less closely applied to the dorsal cortex by stage 6. It no longer
makes contact with the proximal ends of trichocysts along most of its length and most
of the intranuclear microtubules have disassembled; very few microtubule profiles
were detected (Table 1).
DISCUSSION
The presence of longitudinally oriented microtubules within, or around, ciliate
macronuclei as they elongate and divide is well established (see Introduction).
Furthermore, macronuclear elongation is reduced in Paramecium (Ruiz et al. 1976),
Blepharisma (Jenkins, 1977), and Tetrahymena (Tamura, Tsuruhara & Watanabe,
1969; Williams & Williams, 1976) when dividing organisms are treated with colchicine
which induces microtubule disassembly. In some ciliates considerable numbers of
cytoplasmic microtubules are closely applied to the outsides of the envelopes of
elongating macronuclei (Roth & Shigenaka, 1964; Inaba & Kudo, 1972; Paulin &
Brooks, 1975; Jenkins, 1977). This is not the case for P. tetraurelia where the situation
resembles that which occurs during the elongation of several types of animal and
plant cells and certain cell extensions. In all these cases microtubules become oriented
inside, and parallel to the axis of elongation of, a membrane-bound sac. Although
there is no doubt that oriented microtubules are required during the elongation of
certain cells, cell extensions and nuclei, their exact cytoskeletal contribution remains
to be ascertained. It is probably achieved by one of 2 basic mechanisms; active
microtubule sliding or active propulsion of other components alongside tubules
(Tucker, 1979). There are indications that both mechanisms may operate in the
dividing macronucleus of P. tetraurelia. The intertubule linkage and overlapping
arrangement of longitudinally oriented microtubules in the peripheral meshwork, and
the decrease in the number of tubule profiles per nuclear cross-section, as the nucleus
Macronuclear microtubules
149
elongates and becomes thinner, are both compatible with force generation by active
microtubule sliding interactions. So is the rapid elongation rate (40 /tm min"1)
during the transition from stage 3 to 4, which is considerably greater than the fastest
elongation (19 fim min -1 ) reported so far for microtubule bundles assembling in vivo
(Ockleford & Tucker, 1973). The production of lateral surface projections, each of
which contains a compact bundle of microtubules, also indicates that nucleoplasmic
microtubule bundles can transmit compressive forces along their lengths, exert a
pushing action, and move longitudinally relative to the surrounding nucleoplasm. A
pushing action mediated by microtubule sliding interactions could be augmented by
the nucleoli that are attached to the tubules. However, the long stretches of granular
coating material around tubules are almost certainly derived from the cortices of
nucleoli. This indicates that the granules and nucleoli may be propelled alongside
microtubules by elements bound to the surfaces of microtubule walls.
The nuclear envelope probably does not contribute to active force production in any
marked degree during macronuclear division. The formation of both lateral projections
and dorsal pleats indicates that the nuclear envelope has little mechanical strength
and is readily deformed and stretched in regions where it makes contact with rigid
structures such as microtubule bundles and trichocysts. Some of the trichocystassociated deformation may be a consequence of a certain amount of swelling and
partial trichocyst-discharge that occurs during fixation for electron microsopy (Jurand
& Selman, 1969). However, this phenomenon cannot entirely account for the production of longitudinally oriented pleats that become more pronounced as macronuclear
elongation proceeds. It is reasonable to suppose that the pleats are mainly generated
by a 'gouging-action' of the proximal ends of trichocysts (which are firmly anchored
to the pellicle at their distal tips) as the dorsal surface of the elongating macronuclear
envelope slides past them.
Dorsal pleating also gives some indication of the substantial nature and intimacy
of the association between the elongating macronucleus and the dorsal cortex. This
association is perhaps required to ensure medial positioning of the nucleus with respect
to the cleavage furrow and for control of synchrony between macronuclear and cortical
events. Spatial information may be provided by the polarized fine-structural architecture of the cortex (Sonneborn, 1970). In some respects the association may be
similar to that between the cortex and dividing micronucleus in Tetrahymena thermophila (Jaeckel-Williams, 1978). Establishment of the association coincides temporally
with, and perhaps triggers, the adoption of an elliptical shape by the macronucleus.
The marginal microtubule band is probably involved in the production and maintenance of elliptical nuclear shaping along the lines proposed for the cell-shaping
action of the marginal bands of certain elliptically shaped vertebrate blood cells
(Behnke, 1970a, b; Cohen, 1978). There is a positive correlation between the number
of marginal band microtubules and the size of vertebrate erythrocytes (GoniakowskaWitalinska & Witalinski, 1976). The elliptical stage 3 macronucleus does not correlate with the erythrocyte values; it has about one sixth of the number of microtubules
in its band needed to give a reasonable correlation. This is perhaps to be expected.
The mechanics of shape maintenance are likely to be less demanding for a nucleus
150
J. B. Tucker, J. Beisson, D. L. J. Roche andj. Cohen
inside Paramecium than they are for an erythrocyte in, for example, a vertebrate blood
capillary.
It is clear from this examination of microtubule positioning and nuclear shaping
that macronuclear division in P. tetraurelia is definitely not a simple constriction into
2 halves. Hence, it does not fall within the general definition of an amitotic division
(see Introduction). It is, however, still not clear whether the intranuclear microtubules
contribute in any way to regular segregation of chromosomal material. Indeed, the
general procedure by which reasonably balanced genomes are maintained in ciliate
macronuclei over the course of numerous divisions is unresolved (see Sonneborn,
1974). There are some indications that the microtubules may make a contribution.
Fibrils, which are probably microtubules, penetrate some of the nucleoli and chromatin
bodies in the dividing macronuclei of Euplotes eurystomus (Ruffolo, 1978). This study of
P. tetraurelia establishes that some nucleoli are attached to microtubules during
macronuclear division, but no attachments to chromatin were detected. There are
indications that portions of chromatin are attached to nucleoli in the macronuclei of
some ciliates (Raikov & Kovaleva, 1978). This raises the possibility that microtubule/
nucleolus associations may form part of a mechanism for segregation of chromosomal
material during macronuclear division. It has been suggested that attachment of
chromatin to sites on the nuclear envelopes of dividing macronuclei increases the
probability of equal segregation of chromatin in certain hypotrichous ciliates (Walker
& Goode, 1976).
We thank Simone Grandchamp for permitting us to include some of her unpublished data
on the nuclear cycle in Fig. 1. Support from the Science Research Council (U.K.) to J.B.T.,
from the Delegation Generale a la Recherche Scientifique et Technique (grant no. 77-70267)
to J.B., and a training fellowship from the Ligue Nationale Francaise contre le Cancer to J. C.
are gratefully acknowledged.
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{Received 5 February 1980)