J. Cell Sci. 31, 25-35 (i978)
Printed in Great Britain © Company of Biologists Limited 1978
25
THE ULTRASTRUCTURE OF CELL DIVISION
IN EUGLENA GRACILIS
MARCELLE A. GILLOTT AND
RICHARD E. TRIEMER
Department of Botany, Rutgers University, New Brunswick, New Jersey,
08903
U.S.A.
SUMMARY
The ultrastructure of mitosis in Euglena gracilis was investigated. At preprophase the
nucleus migrates anteriorly and associates with the basal bodies. Flagella and basal bodies
replicate at preprophase. Cells retain motility throughout division. The reservoir and the
prophase nucleus elongate perpendicular to the incipient cleavage furrow. One basal body pair
surrounded by a ribosome-free zone is found at each of the nuclear poles. The spindle forms
within the intact nuclear envelope. Polar fenestrae are absent. At metaphase, the endosome is
elongated from pole to pole, and chromosomes are loosely arranged in the equatorial region.
Distinct, trilayered kinetochores are present. Spindle elongates as chromosomes migrate to the
poles forming a dumb-bell shaped nucleus by telophase. Daughter nuclei are formed by constriction of the nuclear envelope. Cytokinesis is accomplished by furrowing. Cell division in
Euglena is compared with that of certain other algae.
INTRODUCTION
Euglena is a unicellular alga which can be grown under a variety of culture conditions. It can be easily manipulated in the laboratory and several mutant strains are
available. These attributes make it an ideal subject for study and over 2300 papers
have been published on Euglena since 1970. Nonetheless, many questions about this
organism remain unanswered.
The euglenoid flagellates have long been recognized as a distinct group of organisms.
They were awarded class status by Fritsch (1935) and constitute either a separate class
or division in most modern taxonomic schemes. The interphase nucleus, with
condensed chromatin, and the distinctive mitotic mechanism are often included in
lists of peculiar euglenoid characteristics.
Leedale (1967, 1968) reviewed the various theories concerning euglenoid mitosis
based on light-microscopic observations dating back to 1895. In addition, some
preliminary observations on the ultrastructure of mitosis in Euglena gracilis are
presented in his latter report. He summarizes the evidence for and against the operation of a typical spindle apparatus in Euglena. The present study will resolve some of
the problems discussed by Leedale by presenting some previously unreported features
of mitosis in E. gracilis.
26
M. A. Gillott and R. E. Triemer
MATERIALS AND METHODS
Euglena gracilis (Klebs) strain Z (Pringsheim) was grown photoheterotrophically on modified
Hutner's media (Vasconcelos et al. 1971). Cultures were placed on a shaker under continuous
light (1500 lux) at 20 °C.
Concentrated cells were fixed in 2 % glutaraldehyde plus 0-5 % neutralized tannic acid
(pH 7-0) made up in Euglena growth media. Cells remained in fixative for 2 h in the dark at 4 CC.
They were then washed 3 times in cold growth media before postfixation. Cells were incubated
in 2 % osmium tetroxide made up in growth media for 2 h in the dark at 4 °C. This was followed
by a rinse in distilled water. Samples were then dehydrated rapidly in a graded ethanol series,
followed by 2 changes of 100 % acetone, and embedded in Epon.
Silver sections were cut on a Sorvall MT 2-B ultramicrotome using a Dupont diamond knife.
After poststaining with uranyl acetate and lead citrate, sections were examined on a Siemens
Elmiskop iA electron microscope at 80 kV.
Abbreviations on
b
c
cl
cv
e
f
g
k
m
figures
basal body
chromosome
chloroplast
contractile vacuole
endosome
flagellum
Golgi
kinetochore
mitochondrion
ms
mt
mastigoneme
microtubule
n
nucleus
"g nuclear granule
np nuclear pore
P pellicle
pa paramylon
r
reservoir
cf cleavage furrow
OBSERVATIONS
Euglena gracilis possesses a single emergent flagellum arising from one of the pair of
basal bodies located at the base of the reservoir. The cells contain large amounts of
paramylon when grown under high light conditions; diskoid chloroplasts and mitochondria are scattered throughout the cytoplasm (Fig. 1). In non-dividing cells, the
pellicle appears in cross-section as a regular series of ridges and grooves (Fig. 1). The
interphase nucleus is centrally located and chromatin remains condensed (Fig. 1)
throughout the cell cycle. Few ribosomes are associated with the outer nuclear
envelope in both non-dividing (Fig. 1) and dividing cells (Figs. 2-10).
Prior to mitosis, the pellicle begins to replicate, as evidenced by the production of
small, newly formed strips alternating with the larger, fully formed pellicle strips
Fig. i. Interphase cell. Note the centrally located nucleus with its condensed
chromatin. Numerous paramylon granules, mitochondria, and several diskoid
chloroplasts are visible. A grazing section of reservoir can be seen in the upper right,
x 3800.
Fig. 2. Preprophase. The nucleus has migrated to the anterior of the cell and becomes
associated with the basal bodies. Note the ribosome-free zone surrounding the basal
bodies, and the tubular nature of the Golgi secreting face (arrowheads). The pellicle
has begun to replicate as evidenced by the small protrusions (arrows) alternating with
the larger, fully formed pellicle strips, x 7500.
Fig. 3. Basal body replication. Basal body replication has occurred, as indicated by the
presence of 4 flagellar cross-sections in the reservoir. Microtubules are present in the
nucleus, x 7000.
Ultrastructure of cell division in Euglena
A
M. A. Gillott and R. E. Triemet
Fig. 4A, B. Prophase. Serial sections demonstrating the parallel elongation of the
reservoir and nucleus. A basal body pair with associated ribosome-free zone is present
at each pole. The polar region of the nucleus is highly invaginated. Note the nuclear
pores, cytoplasmic microtubules and mastigonemes in the reservoir, x 11200.
Ultrastructure of cell division in Euglena
29
(Fig. 2). These continue to develop during division (Figs. 2, 8, 10). A second cytoplasmic indicator of mitosis is increased Golgi activity, indicated by the extensive
tubular proliferation occurring at the secreting face (Fig. 2). Preprophase is characterized by the anterior migration of the nucleus; the nucleus curves around the basal
body pair and is separated from it by a ribosome-free zone (Fig. 2).
Mierotubules begin to form within the intact nuclear envelope at prophase (Fig. 3).
The flagella and their basal bodies have replicated, as indicated by the presence of 4
flagellar cross-sections in the reservoir (Fig. 3). During prophase, the reservoir and
the nucleus elongate parallel to each other and perpendicular to the incipient cleavage
plane (Fig. 4A, B). The basal body pairs migrate to opposite ends of the expanding
reservoir and associate with the poles of the elongated prophase nucleus (Fig. 4 A, B).
Ribosome-free zones remain associated with the basal bodies at the poles (Figs. 4A,
B, 6, 10). The invaginations of the polar regions of the nucleus formed at prophase
persist throughout the division cycle (Figs. 4A, B, 6, 7). Serial sections have failed to
demonstrate the presence of polar fenestrae.
At metaphase, the chromosomes are loosely arranged in the equatorial region
(Figs. 5, 6). Kinetochores, which can be observed at this stage, consist of 2 dense
layers separated by a light layer (Fig. 5 and inset). The mierotubules attach to the outer
dense layer of the trilayered kinetochore (Fig. 5 and inset). The inner dense layer is
closely associated with a less-condensed region of the chromosome (Fig. 5 and inset).
Clusters of dense granules appear in the nucleus (Figs. 5, 7, 8). The endosome enlarges
and elongates from pole to pole by late metaphase (Fig. 6). Although mierotubules
appear to be closely associated with the endosome (Figs. 6—9), no kinetochore-type
structure has been found.
The endosome assumes a dumb-bell shape as the chromosomes move toward the
poles during late anaphase (Fig. 7). Both continuous and chromosomal microtubules
are present. As the chromosomes approach the poles, the nucleus begins to constrict in
the central region, marking the beginning of telophase (Fig. 8). The close association
of the endosome with the central spindle fibres can be seen (Fig. 8). Elongation of the
reservoir parallels that of the nucleus (Fig. 8). By late telophase the nuclear envelope
has constricted further, forming a dumb-bell shape (Fig. 9). The endosome remains
extended through the isthmus until the completion of telophase and the resultant
formation of 2 daughter nuclei.
Cytokinesis proceeds from the anterior of the cell between the 2 daughter reservoirs.
Light-microscopic observation of living E. gracilis cells reveals vigorous metaboly
during cytokinesis. Organelles can be seen to flow freely between the separating
daughter cells. The nuclei remain closely associated with the basal bodies throughout
this process (Fig. 10). Each daughter cell receives a portion of the newly synthesized
pellicle, indicated by the presence of large and small ridges (Fig. 10).
C EL 31
M. A. Gillott and R. E. Triemer
^T1
Ultrastructure of cell division in Euglena
31
DISCUSSION
In certain algae, e.g. Chlamydomonas (Triemer & Brown, 1974), and the zoospores
of Ulothrix (Floyd, Stewart & Mattox, 1972 a) and Klebsormidium (Floyd, Stewart &
Mattox, 1972ft),flagellar abscision occurs prior to mitosis. The basal bodies are then
free to migrate to the nucleus and function as centrioles. Euglena, however, retains its
motility throughout the division cycle. Therefore, the anterior migration of the nucleus
is necessary for the basal bodies to assume a centriolar function. Leedale (1968) cites
this anterior nuclear migration as circumstantial evidence for basal bodies functioning
as centrioles during mitosis, but he was unable to find ultrastructural evidence to
support this hypothesis.
This study presents the first evidence for the association of basal body pairs with
the nuclear poles during division in Euglena gradlis. The curvature of the nucleus in
E. gradlis required serial sectioning to demonstrate the presence of basal bodies at
both poles (Fig. 4A, B). Sommer & Blum (1965), studying Astasia longa, a closely
related euglenoid, found a basal body pair associated with only one pole of a dividing
nucleus. Since the nucleus and reservoir exhibit parallel elongation in both E. gradlis
and A. longa, and flagellar replication preceeds mitosis, it is probable that serial
sections of Astasia would also reveal the presence of basal bodies at both poles.
Pyramimonas, a prasinophycean alga, also retains its flagella during division, and the
events of prophase parallel those in the euglenoids (Pearson & Norris, 1975). The
reservoir and nucleus elongate and the replicated basal bodies associate with the poles.
Small densely staining granules, often clustered, appear in both interphase and
dividing nuclei. These nuclear granules have also been observed by Leedale (personal
communication). Their function is unknown.
The nuclear envelope remains intact throughout mitosis. This type of completely
closed spindle has been found in 2 other algae, Vaucheria litorea (Ott & Brown, 1972)
and Chlamydomonas moetvusii (Triemer & Brown, 1974). A closed spindle has also
been reported in several fungi and protozoa (Albugo, Berlin & Bowen, 1964; Blastocladiella, Lessie & Lovett, 1968; Saccharomyces, Robinow & Marak, 1966; Aspergillus,
Robinow & Caten, 1969; Tetrahymena, Elliot, 1963; Blepharisma, Jenkins, 1967; and
Trypanosoma, Vickerman & Preston, 1970). The trypanosomes are similar to the
euglenoids in certain mitotic features. The persistent endosome of T. rhodesiense
becomes ensheathed by the spindle which traverses the centre of the nucleus. The
endosome of E. gradlis is also closely associated with several spindle microtubules. In
trypanosomes, however, the chromosomes are attached to the nuclear envelope and
the basal bodies are not associated with the nucleus (Vickerman & Preston, 1970).
Fig. 5. Metaphase. Section through equatorial region near the periphery of the
nucleus. Chromosomes are loosely arranged on the metaphase plate. Note the
kinetochores and electron-dense granules, x 11400. Inset: Higher magnification of a
trilayered kinetochore (outlined in Fig. 5). x 33000.
Fig. 6. Longitudinal section of nucleus. The endosome is elongated perpendicular to
the metaphase plate. Note the basal body with ribosome-free zone near the nuclear
pole. X8550.
3-2
M. A. Gillott and R. E. Triemer
Vltrastructure of cell division in Euglena
33
Kinetochores, which had not been previously described at the ultrastructural level
in euglenoid flagellates, were observed at metaphase. Using light microscopy, Saito
(1961) had observed ' V - and 'L'-shaped chromosomes during anaphase in E. viridis.
He interpreted these chromosomal configurations as evidence for kinetochores. Since
other authors failed to find evidence of kinetochores (see Leedale, 1968) it was
proposed that chromosomal movement occurred independently of the spindle. The
kinetochores observed in E. gracilis have a trilayered structure similar to that described
in Chlamydomonas (Triemer & Brown, 1974). The existence of typical kinetochores in
E. gracilis provides evidence for the operation of a normal spindle apparatus as the
mechanism controlling chromosomal movements during mitosis.
Observations of the living cells with Nomarski optics indicates the presence of 2
reservoirs during late anaphase-early telophase. The formation of 2 reservoirs during
anaphase has also been observed in E. gracilis by Leedale (1968), and in Astasia longa
(Sommer & Blum, 1965) and Pyramimonas (Pearson & Norris, 1975). Initiation of the
cleavage furrow between the flagella, and therefore between their attached basal
bodies, guarantees the distribution of one basal body pair to each daughter cell.
During cytokinesis, metabolic movements result in the flow of organelles between the
separating cells. Hence, the continued association of the nucleus with the basal bodies
may provide a method of ensuring the passage of one nucleus to each daughter cell.
The presence of microtubules, as well as their position and orientation during
cleavage (furrowing vs. phragmoplast vs. phycoplast), has been accorded taxonomic
and phylogenetic significance (Pickett-Heaps, 19726, 1975; Stewart, Mattox & Floyd,
1973). Simple furrowing has been observed in Ulva (Lovlie & Braten, 1968), Trichosarcina and PseudendocIonium (Mattox & Stewart, 1974), and in Pyramimonas (Pearson
& Norris, 1975). A few algae utilize phragmoplasts (Pickett-Heaps, 1967; Fowke &
Pickett-Heaps, 1969; Marchant & Pickett-Heaps, 1973), while many of the other algae
employ phycoplasts (Marchant, 1977; Pickett-Heaps, 1972a, 1975; Triemer & Brown,
1974)Neither phragmoplasts nor phycoplasts are found in Euglena and microtubules are
not associated with the cleavage furrow. The twisting motions observed during
cytokinesis with the light microscope lends support to the hypothesis that the cleavage
furrow in Euglena follows the helical 'line' of one of the pellicle strips (Leedale, 1967;
Hofmann & Bouck, 1976).
The results of our study indicate that Euglena possesses a typical mitotic spindle;
basal body pairs are associated with the poles, and kinetochores attach the chromosomes to spindle fibres. The endosomal elongation appears to be guided by the spindle
Fig. 7. Anaphase. Chromosomes have moved toward the poles. The endosome assumes
a dumb-bell shape, x 9450.
Fig. 8. Early telophase. Oblique section of the elongated endosome with associated
microtubules. An interzonal spindle is present. Note the elongated reservoir parallel
to the nucleus, x 7750.
Fig. 9. Telophase. Daughter nuclei beginning to reform. The elongated endosome
still spans the isthmus, x 8400.
M. A. Gillott and R. E. Triemer
tio
Fig* 10. Cytokinesis. Daughter nuclei remain associated with the basal bodies until the
completion of cleavage, x 6500.
fibres, which may attach directly to the endosome. The strongest remaining argument
against the operation of a normal spindle apparatus is the lack of inhibition by
colchicine. Leedale (1968) suggests that the failure of colchicine to inhibit chromosomal movements is due to its inability to enter the cell. Studies by Silverman &
Hikida (1976) on pellicle microtubules also suggest that colchicine may not penetrate
the pellicle complex.
This work was supported in part by a Biomedical Research Support Grant and a Research
Council Grant from Rutgers University. We also wish to acknowledge the Bureau of Biological
Research for providing the electron-microscopy facilities used in this research.
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{Received 15 August 1977)