Journal of Electron Microscopy 60(Supplement 1): S117–S136 (2011)

doi: 10.1093/jmicro/dfr034

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60th Anniversary Issue: Biological

The cell cycle, including the mitotic cycle and organelle
division cycles, as revealed by cytological observations
Yuuta Imoto, Yamato Yoshida, Fumi Yagisawa, Haruko Kuroiwa
and Tsuneyoshi Kuroiwa*
Research Information Center for Extremophiles, Graduate School of Sciences, Rikkyo University,
3-34-1 Nishiikebukuro, Toshimaku, Tokyo 171-0825, Japan
*To whom correspondence should be addressed. E-mail: tsune@rikkyo.ne.jp, 09ld004f@rikkyo.ac.jp

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Abstract It is generally believed that the cell cycle consists essentially of the
mitotic cycle, which involves mitosis and cytokinesis. These processes

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are becoming increasingly well understood at the molecular level.
However, successful cell reproduction requires duplication and segre-
gation (inheritance) of all of the cellular contents, including not only the
cell-nuclear genome but also intracellular organelles. Eukaryotic cells
contain at least three types of double membrane-bounded organelles (cell
nucleus, mitochondria and plastids), four types of single membrane-
bounded organelles (endoplasmic reticulum, Golgi apparatus, lysosomes
and microbodies) and the cytoskeleton, which comprises tubulin-based
structures (including microtubules, centrosome and spindle) and actin
microfilaments. These membrane-bounded organelles cannot be formed
de novo and daughter organelles must be inherited from parent organelles
during cell cycle. Regulation of organelle division and its coordination
with the progression of the cell cycle involves a sequence of events that
are subjected to precise spatio-temporal control. Considering that the
cells of higher animals and plants contain many organelles which tend to
behave somewhat randomly, there is little information concerning the div-
ision and inheritance of these double- and single-membrane-bounded
organelles during the cell cycle. Here, we summarize the current cytologi-
cal and morphological knowledge of the cell cycle, including the division
cycles of seven membrane-bounded and some non-membrane-bounded
organelles. The underlying mechanisms and the biological relevance of
these processes are discussed, particularly with respect to cells of the
primitive alga Cyanidioschyzon merolae that have a minimum of orga-
nelles. We discuss unsolved problems and future perspectives opened by
recent studies.
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Keywords cell cycle, organelle-division cycle, mitochondrial-division cycle,
chloroplast-division cycle, microbody-division cycle, Golgi apparatus-div-
ision cycle
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Received 17 January 2011, accepted 19 April 2011
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Introduction of the cell cycle are generally understood to corre-

Cells reproduce by duplication of their contents spond to the mitotic cycle. Using autoradiography,
and subsequent division. This cycle of duplication Howard and Pelc [1] discovered that nuclear DNA is
and division is called the cell cycle, and the events synthesized during a stage of interphase designated

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© The Author 2011. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved.
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S118 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
as S-phase. The S phase is followed by a gap (G2
phase), nuclear and cell division (M phase) and a
second gap (G1 phase) before the next S phase.
The functions of these phases have been identified
in many cells, including human and yeast cells [2,3].
It is generally believed that the structural and func-
tional aspects of the cell cycle are primarily associ-
ated with the cell nucleus. However, the cell also
contains organelles enclosed by double membranes
(mitochondria and plastids) and by a single mem-
brane (endoplasmic reticulum (ER), dictyosomes of
the Golgi apparatus and lysosomes or vacuoles).
Clearly, successful cell reproduction requires faith-
ful duplication and segregation of all of the cellular
contents, which includes the intracellular organelles
as well as the nuclear genome. Mitochondria and
plastids are regarded as descendants of endosym-
biotic prokaryotes. They have their own DNA and
proliferate by division. Regulation of organelle div-
ision and its coordination with the progression of
the cell cycle involves a sequence of events that are
subjected to a precise spatio-temporal control. To
understand the cell cycle, we need to recognize
coordination between the mitotic cycle and orga-
nelle division cycles. Therefore, it is important to
survey the division cycle of double- and
single-membrane-bounded organelles and the
dynamics of cytoskeleton during the cell cycle.
We have proposed the concept of the ‘mitochon-
drial division (MD) cycle’ analogous to the mitotic
cycle [4–6]. In the plasmodium of the slime mold
Physarum polycephalum, mitosis and the mitotic
cycle are naturally synchronized and, in addition,
the mitochondria contain large rod-shaped com-
plexes of DNA and proteins, termed nucleoids. The
mitotic and MD cycles were precisely examined
using 3H-thymidine autoradiography and it was
shown that mitochondrial phases of the division
cycle, i.e. mt-S, mt-G2, mt-M and mt-G1 lasted 7.7,
2, 1.5 and 3 h, respectively, while the mitotic S, G2
and M phases lasted 6, 7 and 1 h, respectively. G1
lasted for a very short time. Although the timing of
the phases of the mitotic and MD cycles differed
markedly, the total generation time of the mitotic
cycle (14 h) was the same as that of mitochondria
(Fig. 1).
The plastid division (PD) cycle was examined in
tobacco Bright Yellow 2 (BY-2) cells using
cytological techniques. The plastid DNA synthesis
phase ( pt-S) during the cell cycle of cultured BY-2
cells was examined by 3H-thymidine autoradiog-
raphy following medium renewal. The timing of the
pt-S phase differed from that of the mitotic S phase.
These observations suggest that the PD cycle pro-
ceeded independently from the mitotic cycle [7].
Plastid and mitochondrial DNA syntheses were also
observed in root meristem and cultured BY-2 cells
by immunofluorescence microscopy of Technovit
sections using an antibody against 5-bromodeoxy
uridine (BrdU) and co-fluorescent staining with
DAPI and quantitative Southern hybridization [8]. It
was shown that large quantities of both mitochon-
drial and plastid DNA were preferentially syn-
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thesized prior to S-phase. However, in plants,
animals and fungi, it has been difficult to determine
the timing of each phase of the division cycle of
double-membrane-bounded organelles. However,
the difference in the timing of the organelle S-phase
and mitotic S-phase has been established.
Partitioning or division (inheritance) of single-
mem-brane-bounded organelles occurs in many
organisms. Additionally, the dynamics of cyto-
skeletal proteins and of centrosomes, which play
major roles in the cell transport system and in cell
division during the mitotic cycle, must be examined
during the cell cycle [2,3]. During G1 phase, the two
Fig. 1 Diagram of the mitotic cycle of P. polycephalum. The cycle
proceeds clockwise. The outer circle represents mitochondrial events
and the inner circle depicts cell nuclear events. The duration of each
phase is shown in hours. This figure was adapted from Ref. [5].

orthogonal centrosomes separate. In general, in
these multi-organelle organisms, it has been difficult
to demonstrate each division or partitioning of
double-and single-membrane-bounded organelles
because the cells contain large numbers of these
organelles and divisions or partitioning occur more
or less randomly (mitochondrial and plastid fusion
may also occur).
The unicellular red alga Cyanidioschyzon
merolae is a very small organism (1.5–2.0 μm diam-
eter), whose cells offer unique advantages for
studies of double- and single-membrane-bounded
organelle division cycles. They contain a minimal
set of organelles; i.e. a cell nucleus, a mitochon-
drion, a chloroplast, a microbody, an outer nuclear
membrane/ER, a single Golgi apparatus and a few
lysosomes [9–11]. Its organelle divisions can be
highly synchronized with the light/dark cycle. The
genome of C. merolae has been completely
sequenced and it is one of the smallest genomes
(16.5 Mb), with the lowest number of genes among
free-living eukaryotes [9,12]. Because most of the
genes are present in low copy numbers and lack
introns, they are suitable for identifying functioning
genes by proteome and transcriptome analysis.
Moreover, microarray analysis of its gene
expression profile during organelle division and
inheritance was recently reported [13].
Besides studies of the mitotic cycle, MD and PD
cycles have been examined by genome analysis
[9,12], matrix assisted laser desorption ionization
time of flight mass spectrometry (MALDI-TOF-MS)
[14–17] and gene targeting [15–19]. The myosin gene
was absent from the genome [9,12] and actin genes
were not expressed. Therefore, microfilaments and
intermediate filaments could not be identified
throughout cell cycle by transcriptome and pro-
teome analyses [9,13]. In addition, during G1 phase,
cytoskeletal microtubules are absent [20].
Miyagishima et al. [21] presented time-lapse video
images of C. merolae during the M phase under
phase contrast light microscopy. The images were
taken at 20 min intervals for the first 220 min after
the onset of the chloroplast division, and at 10 min
intervals, starting 340 min after the onset of the
chloroplast division [21]. The temporal sequence of
electron microscopic images of the division process
of double- and single-membrane-bounded
Y. Imoto et al. Cell cycle and organelle division cycles S119
organelles, and of non-membrane-bounded orga-
nelles, during cell cycle could be inferred from the
images of the chloroplasts in living cells.
Here, we review the current cytological and mor-
phological knowledge concerning the cell cycle,
including the division cycles of seven
membrane-bounded-organelles (cell nucleus,
mitochondria, chloroplasts, microbodies, lysosomes,
the Golgi apparatus and ER) and non-memb-rane-
bounded organelles (centrosomes and spindle). We
discuss the underlying mechanisms and the biologi-
cal relevance of this process, particularly in C.
merolae cells in comparison with other organisms
(Fig. 2). Furthermore, we discuss unsolved funda-
mental problems of the cell cycle related to the
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mitotic and organelle division cycles and future per-
spectives opened up by recent studies.
Division cycle and inheritance of
double-membrane-bounded organelles
(cell nuclei, mitochondria and plastids)
Mitotic cycle
The typical eukaryotic cell cycle is divided into G1,
S, G2 and M phases [2,3]. The generation times and
the durations of each phase have been determined
in many organisms, for example, the generation
time of the plant Crepis capillaries is 10.4 h, com-
prising the DNA synthesis phase (S phase 5.1 h),
the gap between S phase and M phase (G2 phase
2.2 h), mitosis (M phase 1.5 h) and the gap between
M and S phases (G1 phase 1.6 h) [22,23]. The con-
densed mitotic chromosomes are readily visible in
almost all cells of Bikonta, Opisthokonta and
Amoebozoa [2,3]. In the case of budding yeasts, the
16 condensed meiotic chromosomes are readily
visible [24], but are less easily seen in mitosis [2,3].
The cytological mechanisms for condensation of
chromosomes from interphase to mitosis have been
examined in detail in plants using high-resolution
electron microautoradiography [24–27] and the mol-
ecular mechanism of chromosome condensation in
animals have been summarized by Hirano [28]. The
cell divides by forming a partition and splitting in
two. Cytokinesis occurs by actin contractile rings
[2,3]. In contrast to the situation in higher eukary-
otic cells, the nuclear envelop of the yeast cell does
not break down during M phase. The microtubules
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Fig. 2 Summary of the division cycles and the temporal relationships of the three double-membrane-bounded organelles (cell nucleus,
mitochondrion and chloroplast ( plastid)) and the four single-membrane-bounded organelles (ER, Golgi apparatus, lysosome and microbody)
and centrosome in Cyanidioschyzon merolae cells. Each C. merolae cell has a minimum set of organelles comprising one cell nucleus, one
mitochondrion and one chloroplast, simple ER, one Golgi apparatus, one microbody and a few lysosomes. The organelle divisions can be
highly synchronized by the light/dark cycle. Chloroplast, mitochondrion and cell nucleus divisions occur in that order. Mitotic chromosomes
are not organized at M phase. Chloroplast and mitochondrion divide using the chloroplast (PD ring) and MD machineries (MD ring),
respectively. Microbody and lysosome are inherited using the mitochondrion as a carrier. Tubulins are absent in G1 phase, synthesized as a
monomer in S and organized into mitochondrial spindle and mitotic spindles in M phase. Mitotic cycle: G1, S, G2, M; MD cycle:
mitochondrial G1 phase (mt-G1), mitochondrial S phase (mt-S), mitochondrial mitotic phase (mt-M); and plastid cell cycle: plastidG1 phase
(pt-G1), plastid S phase ( pt-S), plastid division phase ( pt-M), the division cycles of the ER, Golgi apparatus, lysosome, microbody, and the
non-membrane-bounded organelle (centrosome). Double arrowheads indicate the time point of the organelle replication and single
arrowheads indicate the time point of the organelle division. Time shows hours after the second cell cycle.

of the mitotic spindle form inside the nucleus and
are attached to the spindle pole bodies at its
periphery.
Currently, it appears that the cell cycle is com-
posed of a mitotic cycle, a MD cycle, a PD cycle, a
microbody division cycle, a lysosome (vacuole) div-
ision cycle, a Golgi apparatus division cycle and an
ER division cycle. Organelle division in C. merolae
can be highly synchronized by a light/dark cycle
and, under those conditions, the divisions of Golgi
apparatus, chloroplast, mitochondria, microbody,
lysosomes, cell-nucleus, ER and finally cytokinesis
occur in that order. Based on studies of slime
molds and higher plants, it has been suggested that
the cell cycle is composed of a mitotic cycle, a MD
cycle and a PD cycle.
In 1994, Suzuki et al. [29] examined the coopera-
tive behavior of double-membrane-bounded orga-
nelles (cell nuclei, mitochondria and plastids)
during the cell cycle of C. merolae by epifluores-
cence microscopy and fluorometry after staining
with DAPI, using a video-intensified microscope
photon-counting system (VIMPCS). The mitochon-
drial DNA and the chloroplast DNA were syn-
thesized during stage I (G1 phase), while the
cell-nuclear DNA was duplicated in stage II
(S phase). The mitochondrial and chloroplast div-
isions began simultaneously in stage II, and chloro-
plast division finished just prior to MD. The
mitochondrial and chloroplast-nuclear divisions
were completed in stage IV (M phase). The gener-
ation time of this cycle was 28 h. They were able to
identify the duration of each phase of the cell
cycles (G1 phase, 19 h; S phase, 6 h; G2 phase, 2 h;
M phase, 1 h). On the basis of these data, Itoh et al.
[30,31] suggested relationships between the mitotic
cycle and chloroplast, mitochondrial, and micro-
body division cycles in C. merolae using electron
microscopy. However, they could not determine the
timing of each phase of MD and PD cycles.
To determine the timing of each of the G1, S, G2
and M phases of the cell nuclear divisions, and of
the mitochondrial (mt-G1, mt-S, mt-G2 and mt-M)
and plastid ( pt-G1, pt-S, pt-G2 and pt-M) phases
during cell cycle, Imoto et al. [20] measured the
DNA content of each cell nucleus, mt-nucleus
(mt-nucleoid) and pt-nucleus ( pt-nucleoid) directly
using an improved VIMPCS method, after staining
Y. Imoto et al. Cell cycle and organelle division cycles S121
with DAPI. The results were consistent with the
previous data. The pt-S phase of the PD cycle and
the mt-S phase of the MD cycle always preceded
the S phase of the mitotic cycle by approximately
1 h. The division of the plastid was completed first,
followed by the mitochondria, and then, finally, by
the cell nuclei. The durations of each phase of the
MD (mt-G1, 12 h; mt-S 1 h; mt-G2, 4 h; mt-M, 2 h)
and PD cycles ( pt-G1, 14 h; pt-S, 1 h; pt-G2, 2 h;
pt-M, 2 h) could be estimated. These observations
clearly demonstrated that the cell cycle should be
understood not only in terms of the mitotic cycle,
but also in terms of MD and PD cycles.
These data indicate that the cell cycle is com-
posed of mitotic, MD and PD cycles, which appear
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to proceed independently of each other. In
C. merolae, chloroplasts, mitochondria and cell
nuclei divided in that order, while in the primitive
red alga Cyanidium caldarium, the sequence was
chloroplasts, cell nuclei and mitochondria [32,33].
Therefore, there a mechanism that determines the
order of chloroplasts, cell nuclei and MDs must be
present. Moreover, in multi-organelle organisms
such as Amoebozoa, Biokonta and Opithsokonta,
the mechanism that determines the order of orga-
nelle divisions must be even more complex.
A feature of cell-nuclear behavior observed
during the G1 phase of C. merolae was that the
lower end of the cell-nucleus protruded at the
center of the mitochondrion, which in turn became
doughnut-shaped [20]. In this cell, the cell-nucleus
possessed a single central nucleolus and associated
chromatins, which consisted of a set of three homo-
geneous copies of the ribosomal gene and one
histone gene. At this stage, a cytoskeleton and cyto-
skeletal proteins (actin, 10 nm filaments, tubulin)
were absent in cells of C. merolae although cells at
G1 phase (10 h) moved actively as amoebae.
Therefore, the dynamic changes exhibited by intra-
cellular organelles must have been performed
without a cytoskeleton. During the S phase, the
lower end of cell-nucleus was taken out so that the
cell-nucleus containing the nucleolus became
spherical. Tubulin molecules were synthesized as
monomers and they diffused throughout the entire
cytoplasm for about 1.5 h during the S phase.
Probably, the single mother centrosome core
(without microtubules) was formed in the
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cytoplasm near the peripheral end of the mitochon-
drion during early S phase and it was replicated
and divided into two daughter centrosome cores
during late S phase (unpublished data). For 1 h
during the G2 phase, the cell nucleus became
football-shaped. Microtubules were focused on two
daughter centrosome cores and formed centro-
somes. For 2 h during the early M phase, the
nucleus changed from a football-like structure into
a Napoleon hat-like configuration (inverted cup) of
which the two ends were associated with the cen-
trosomes. Several microtubules extended from the
centrosomes and were positioned on the mitochon-
drion to form a mitochondrial spindle. The nucleo-
lus began to move from the central region of the
cell nucleus to the upper region. For 2 h, during the
middle M phase, the cell nucleus became peanut-
shaped and homogenous. During metaphase, rod-
shaped mitotic chromosomes were never observed
although condensins (multi-subunit protein
complex that play a central role in mitotic chromo-
some assembly and segregation) [28] were present
and a mitotic nuclear spindle was formed between
daughter centrosomes. Consequently, chromosome
condensation did not occur in C. merolae although
CENH3-containing kinetochores reconstituted into
two separate dot and co-localized with centrosomes
during early metaphase and drew uncondensed
regions of the chromosomal arms into the daughter
cell nuclei [34] (Fig. 3a). For 2 h during late M
phase, the dumbbell-shaped cell nucleus divided
into daughter cell nuclei by the activity of mitotic
spindle. The central spindle then pushed the daugh-
ter cell nuclei apart from the daughter cells. The
daughter nuclei then changed back from being cup-
shaped to spherical and cytokinesis occurred in the
absence of an actin cytoskeleton [9,32]. After the
mitotic division, the cell-nucleus with its central
nucleolus resumed a spherical shape, suggesting
that a part of cell nucleus did not protrude into
mitochondrion.
Aspects of C. merolae cell division differ from
that observed in other cells and are summarized as
follows: (a) Tubulin synthesis is confined to a single
phase, and tubulin and actin do not exist during G1
phase; (b) A mitochondrial spindle is present; (c)
Condensation of chromosomes does not occur
during M phase although proteins from the
condensin family are present; (d) Cytokinesis
occurs in the absence of a contractile rings of actin.
In preliminary experiments, EF1α has been located
in the contractile ring region, and it is likely that
this protein controls cytokinesis in many eukaryotic
cells (Imoto Y, Nishida K, Yagisawa F, Yoshida M,
Yoshida Y, Ohnuma M, Fujiwara T, Kuroiwa H and
Kuroiwa T. unpublished data) (Fig. 3b).
Mitochondrial behavior
As described above, the concept of the organelle
division cycle has been evoked by the MD cycle of
P. polycephalum. Mitochondria have their own
DNA which is organized using basic proteins to
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form organelle nuclei (nucleoids). Organelle nuclei
are universal in eukaryotes and the mechanism of
condensation of mitochondrial nuclei has been
summarized [6,35,36]. Mitochondrial nuclei are syn-
thesized during a specific phase of the MD cycle,
which consists of the mt-S phase, the mt-G2 phase,
the mt-M phase, and the mt-G1 phase. The mt-M
phase is characterized by two main events: division
of the mitochondrial nuclei, which is followed by
division of the matrix (the so-called MD or mito-
chondriokinesis). The mechanism of mitochondrial
nuclear division has been summarized by Kuroiwa
et al. [35].
Mitochondriokinesis occurs after mitochondrial
nuclear division and in this respect it resembles bac-
terial cytokinesis. We have described the fine struc-
ture and dynamics of the MD ring which provide the
morphological bases for mitochondriokinesis
[37,38]. Electron microscopic studies first identified
a small electron-dense ring at the constriction sites
of dividing mitochondria in P. polycephalum and,
subsequently, similar structures have been observed
in Nitella flexilis [6], Nannochloropsis oculata [39]
and higher plants [38]. However, in multi-organelle
organisms such as animal and plants, it was difficult
to examine the timing of each phase of the MD cycle
and of the MD ring because their cells have many
organelles which divide asynchronously, and the
ring is too small to be studied.
As C. merolae cell has a simple disc-shaped mito-
chondrion between a single cell nucleus and a
single chloroplast, it is easy to observe the behavior
of the mitochondrion throughout the cell cycle. The
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Fig. 3 (a) Immunofluorescent micrographs showing the cell nucleus in C. merolae during G2 and M phase. (a–d) Phase contrast
immunofluorescence images of DAPI (blue), CENH3 (green) indicating centromere and α-tubulin (red) in M phase cell. DAPI-stained cell
nucleus (white arrowheads) shows that chromosome condensation did not occur in C. merolae. (e–h) Immunofluorescence images of CENH3
(green) and DAPI (blue) through cell cycle. Dispersed CENH3 signals are focused on centrioles at M phase. (i) Model of the M phase
(metaphase) cell of C. merolae. Scale bar, 2 μm. This figure includes photographs that appeared in Refs [20] (a–d) and [34] (e–h).
(b) Immunofluorescent micrographs showing the early cytokinesis in C. merolae. (a–d) Phase contrast immunofluorescence images of DAPI
(blue), EF1α (green) indicating and α-tubulin (red) in early cytokinesis cell. The signals of EF1α are detected in the contractile ring region
(white double arrow heads).

time course has been deduced from fluorescence
images using the VIMPCS system [20], and from
living cells [21]. The morphological changes of the
organelles were as follows. For 9 h during early and
middle mitotic G1 phase (mitochondrial G1 phase),
the mitochondrion exhibited a discoidal shape,
dented at the center because the cell nucleus pro-
truded into the mitochondrion. During late G1
phase (mt-S phase), as the hollow center of the
mitochondrion became flat again, the nucleus
became disc-shaped, and then quickly elongated.
The mitochondrion synthesized its own DNA for
about 1.5 h. During S (early mt-G2) phase for 2 h,
the mitochondrion extended into an elongated
disc-shaped structure and the microbody attached
to the peripheral end of mitochondrion. For 1 h
during G2 phase (middle mt-M), the MD machinery,
composed of an inner matrix ring and an outer
cytoplasmic ring, was formed at the division site,
immediately after formation of plastid-dividing (PD)
machinery. The microbody then moved from the
end to the central region of mitochondrion and
attached to the outer ring of the MD machinery.
The centrosomes of the mitochondrial spindle were
formed at the each end of mitochondrion. For 2 h
during prophase (late mt-G2), the microbody began
elongating along the MD machinery of the cup-
shaped mitochondrion and surrounded the outer
ring of the MD machinery. The MD machinery then
began to contract at the division site. Nishida et al.
[40–43] examined the structure and function of the
MD ring in detail. The MD machinery was com-
posed of the outer machinery (outer MD ring,
dynamin ring, Mda1 ring) and the inner machinery
(inner MD ring, FtsZ ring). By the contraction of
MD machinery, the mother mitochondrion divided
into two daughter mitochondria. For 2 h during
metaphase (mt-M phase), the dumbbell-shaped
mother microbody divided and separated into
spherical daughter microbodies by the activities of
S124 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
electron-dense patches (50 nm in diameter) at each
side of the microbody, and microtubules of the
mitochondrial spindle. During anaphase (mt-M
phase), each microbody detached from the daugh-
ter mitochondria and mitosis and cytokinesis then
occurred [37,40–43]. Recent interesting progress in
the field is that MD machineries have now been iso-
lated from C. merolae cells [16]. A novel ZapA-like
bacterial protein, ZED, has provided new insights
into MD and the birth of eukaryotic cells.
Bacterial cell division systems that include FtsZ
have been found throughout prokaryotes. Mitochon-
dria arose from an endosymbiotic α-proteobacterial
ancestor and proliferate by division. However, it was
previously unclear how the MD system was estab-
lished from the bacterial division. Bacterial cytokin-
esis occurs by invagination of the cell membrane as
soon as the bacterial nucleus has been replicated.
Filamentous temperature-sensitive (fts) genes for
bacterial cytokinesis were originally identified in
Escherichia coli mutants collected in the late 1960s.
Several proteins involved in bacterial protein recruit-
ment to the division site in an FtsZ-dependent
manner had been identified, including FtsA, ZipA,
FtsK, FtsQ, FtsL, FtsI, FtsN and FtsW [37].
According to the endosymbiotic theory, mitochon-
dria were derived from free-living α-proteobacteria
that became engulfed by eukaryotic host cells.
Mitochondria proliferate on their own during the cell
cycle, in a manner similar to bacterial division.
However, mitochondria have persisted within
eukaryotic cells for a long period—approximately 1–
2 billion years. Since the organelle genome sizes,
even in lower eukaryotes, are less than 10% of the
size of the genomes found in free-living bacteria, it is
thought that following endosymbiosis, initially about
90% of the endosymbiotic bacterial genes were trans-
ferred to the host cell nuclear genome, or lost
entirely. As a consequence, only one FtsZ gene
remains as the mitochondrial dividing gene. Yoshida
et al. [16] have now isolated the MD machinery and
identified a protein ZED that constricts the basal
structure of the MD machinery with FtsZ (Fig. 4).
ZED contains a mitochondrial transit signal and two
coiled-coil regions and has a partial homology with
the bacterial division protein ZapA. In cytological
studies, ZED was observed to accumulate to form a
ring beneath the inner mitochondrial membrane and
to colocalize with FtsZ. ZED proteins are expressed
just before MD. ZED interacted with FtsZ1 to form
the basic structure of the MD machinery and was
required for the MD. Bacterial ZapA and mitochon-
drial ZED were also functionally very similar, which
implies that the bacterial cell division system was
incorporated into the MD machinery during evol-
ution with subsequent loss of most of the bacterial
dividing genes.
Plastid (chloroplast)behavior
The division of plastids (chloroplasts) is universally
present in Biokonta. As mitochondria, plastids
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possess their own DNA which is organized by basic
proteins to form nucleoids ( plastid nuclei) and plas-
tids are believed to be closely associated with the
evolution of eukaryotes [44]. Dynamic changes of
plastid nuclei have been examined during the life
cycle of algae and higher plants and the mechanism
of the condensation of plastid nuclei has been
studied [45]. Plastid DNA (nuclei) is synthesized
during a specific phase of the division cycle. The
phases of the plastid division cycle consist of the
pt-S phase, the pt-G2 phase, the pt-M phase and
the pt-G1 phase. During the pt-M phase, the division
can be separated into two main events: division of
the plastid nuclei and division of the stroma (the
so-called plastid division or plastidkinesis). The
mechanism of plastid nuclear division is summar-
ized below [45].
Plastid kinesis occurs after plastid nuclear div-
ision and is similar to bacterial cytokinesis, based
on the evidence of the FtsZ protein which is
involved in plastid division in Arabidopsis thaliana
[46,47]. The fine structure and dynamics of the
plastid division ring (PD ring) provide the morpho-
logical bases of plastid division [37,38]. Electron
microscopic studies first identified a small
electron-dense ring at the constriction sites of divid-
ing plastids in C. caldarium and subsequent studies
have shown that similar structures are universally
present among the Bikonta [39]. However, in multi-
organelle algae and plants, it is difficult to examine
the timing of each phase of the plastid division
cycle because their organelles divide asynchro-
nously and the ring is too small to be studied.
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Fig. 4 Immunofluorescent and electron microscope images showing ZED rings of mitochondria in C. merolae cells. (a–e) Phase contrast
immunofluorescence images of ZED (green) and mitochondrial matrix (red) through MD. (f–h) Phase contrast immunofluorescence images
show co-localization of ZED (green) and FtsZ (red). (i) Immuno-electron micrographs images of ZED. Immunogold particles (indicating ZED;
black arrowheads) are localized on the matrix side of the MD site. ( j, k) Model of the MD machinery and its component of ZED ring. ZED
interact with FtsZ1 to constitute the basal structure of MD machinery. This figure includes photographs that appeared in reference [16] (a–i), (k).

C. merolae was also employed to study the mor-
phological changes during plastid division, and to
estimate its time course, using fluorescence imaging
and the VIMPCS system [20], in live cells [21]. For
9 h during the early and middle G1 phase ( pt-G1),
the cup-shaped plastid with its central plastid
nucleus became enlarged and, for about 1.5 h during
late G1 ( pt-S), it synthesized DNA. Over 2 h during S
phase ( pt-G2), the plastid enlarged further and
became peanut-shaped. The chloroplast nucleus
then elongated at one end and attached itself to the
upper wall at one end of the chloroplast. Over a
period of 1 h during G2 phase ( pt-M), the plastid
nucleus elongated further and both ends of the
pt-nucleus were attached to the upper wall at oppo-
site ends of the mother plastid. The inner com-
ponents of the plastid division machinery (FtsZ ring
and PD ring) were formed first, followed by the
outer components (outer PD ring and dynamin ring)
[38]. For 2 h during early M phase ( pt-M), the inner
and outer plastid division machinery contracted,
pinching off the plastid at the division site and divid-
ing the mother plastid into two daughter plastids.
Plastid division was completed just before mitotic
S126 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
metaphase. The plastid division machinery associ-
ated with the daughter plastids was released and
finally broken down. MD, microbody division,
mitosis and cytokinesis then follow in that order.
The mechanism of chloroplast division by the PD
machinery has been reviewed by Kuroiwa et al.
[38,44].
Recently, our understanding of the process of
plastid division machinery has been advanced by
isolation of the PD machinery and by identification
of proteins and other components using MALDI–
TOF–MS [14,15]. During chloroplast division, the
FtsZ ring (localized in the stroma), the PD ring
(in the cytoplasm) and the dynamin ring (in the
cytoplasm) were formed in that order. The PD ring
is the main structural component of the PD
machinery and was observed to be a bundle of fine
filaments 5–7 nm in diameter. Two types of
guanosinetriphosphatase, a bacterially derived
FtsZ and a eukaryote-specific dynamin, have been
characterized as PD-machinery-associated pro-
teins. The isolation and analysis of the PD machin-
ery have been achieved and the complete
sequencing of the genome has enabled proteomic
analysis. To detect the components of the PD ring,
Yoshida et al. [15,16] digested isolated PD machi-
neries with various proteases but were unable to
completely decompose them. Therefore, they
searched for hypothetical components that might
constitute the PD ring. Electron-dense deposits
(indicating carbohydrates) appeared on the PD
ring after staining with periodic acid–horseradish
peroxidase (PA-HRP). This suggested that the PD
ring might have a saccharic architecture. To
confirm this, we isolated PD machineries from
dividing cells of C. merolae. Proteomic analysis
identified a protein, PDR1, a glycosyltransferase
which was present in the PD ring and is widely
conserved from red alga to land plants (Fig. 5).
Electron microscopy showed that PDR1 forms a
ring with carbohydrates at the chloroplast-division
site. Fluorometric saccharide ingredient analysis
of purified PD ring filaments showed that only
glucose was included, and down-regulation of
PDR1 impaired chloroplast division. Thus, the
chloroplasts are divided by the PD ring, which is a
bundle of PDR1-mediated polyglucan filaments. As
thePDR-1 gene is universal in Bikonta [15], it is
clearly a key to solving the molecular mechanism
of plastid division.
Division cycles and inheritance of
single-membrane-bounded organelles
(ER, Golgi apparatus, lysosomes and
microbodies)
The inheritance of single-membrane-bounded, and
DNA-less organelles such as ER, Golgi bodies,
vacuoles/lysosomes and microbodies, as well as of
double-membrane-bound organelles such as mito-
chondria and plastids, is an essential feature of
eukaryotic cell division.
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Division cycle and inheritance of ER and Golgi
apparatus related to cell nucleus
The behavior and inheritance of single-mem-brane-
bounded organelles are related to the metabolism of
these organelles. mRNA is synthesized in the cell
nucleus and moves to the ER for translation.
Proteins are transported from ER to Golgi apparatus,
which processes and packages both proteins and
lipids. Subsequently, these processes are aided by
the activities of lysosomes and microbodies.
Therefore, the localization of these organelles is inti-
mately related to their functions established in their
early evolution. In the primitive eukaryotes, the cell-
nuclear division during mitosis is accompanied by
the inheritance of the ER and Golgi apparatus in
association with the mitotic spindle. By contrast, the
mitochondria are required as the dividing carrier of
microbodies and lysosomes which are carried on the
mitochondrial spindle [20]. Therefore, we review
that the inheritances of ER and Golgi apparatus are
in relation to the cell nucleus, while the inheritances
of microbodies and lysosomes are in relation to
mitochondria. Although single-membrane-bounded
organelles do not contain DNA, the division cycles
of these organelles are basically divided into several
phases: a phase of no growth, a phase of growth
(synthesis of contents) and division, a phase of
migration to a carrier, such as a mitochondrion, and
a phase of separation and release from the carrier.
As in many organisms, the cells contains numerous
single-membrane-bounded organelles, it is difficult
to identify each of these phases. By contrast, in cells
of C. merolae cell, which contain a minimum of
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Fig. 5 Immunofluorescent and electron microscopyimages of PDR1 in C. merolae cells. (a–f ) Phase contrast immunofluorescence images of
PDR1 (green) and autofluorescence of plastids (red) through plastid division. (g) Electron microscopy using the PA-HRP method in a cell.
The PD machinery is stained black. Magnified image shows spectral colors correspondingto the intensity of staining signals of the region
around the PD ring (white arrowheads) (see color scales). cp, chloroplast. Scalebar, 2 μm. (h) Immuno-electron microscopy images of PDR1
in chloroplast division. Magnified image of the region around the PD ring (black arrowheads). n, nuclear; mt, mitochondria. Scale bar, 1 μm.
(i, j) Model of PD-machinery and PDR1 function. After the FtsZ ring (blue) is assembled (step 1), PDR1 proteins (red) are recruited at the
chloroplast division site and synthesize F-PDRs (PD ring filaments) (black) from UDP-glucose (step 2a). Other PDR1 proteins bind to
elongated F-PDRs and synthesize more F-PDRs (step 2b and step 3). This figure includes photographs that appeared in Ref. [15].

these organelles, it is possible to determine the
approximate timing of these phases.
The ER and Golgi apparatus are directly con-
cerned with the functions of cell nucleus, their
behavior and their inheritance during cell cycle are,
therefore, intimately connected with those of the
cell nucleus. Sheahan et al. [48] reported that
during the division of the protoplasts of higher
plants, the act in cytoskeleton was required for
balanced inheritance of chloroplasts, mitochondria
and ER. However, the genetic mechanism for the
inheritance of the ER is unclear.
By contrast, the division and inheritance of the
Golgi apparatus (dictyosome) was cytologically
examined in algae. The function of the Golgi appar-
atus is to process and package proteins and lipids,
particularly in the processing of proteins for
secretion. Noguchi [49] reviewed Golgi apparatus
division cycles. In the alga Chlorococcum infusio-
num, the Golgi apparatus was localized close to the
cell nucleus. It was not duplicated during cell wall
formation, but during the binary division of the cell
nucleus [50]. The numerous Golgi apparatuses in
the large unicellular green algae Micrasterias sp.
were located not only around cell nuclei, but also
throughout the cytoplasm [51]. During the mitotic
phase, their numbers increased from about 70 to
150 in Micrasterias pinnatifida, and from 200 to
400 in M. crux-melitensis. These additional Golgi
apparatuses, which were distributed throughout the
entire cytoplasm in M. pinnatifida [52] and
Closterium ehrenbergii [53], appeared synchro-
nously at the premitotic stage. Noguchi [50] con-
cluded that Golgi apparatuses in plant and animal
cells divided during the M phase. The mother
Golgi apparatus divided at the center from the cis
side to the trans side to form daughter Golgi
apparatuses [54]. However, no special structure or
gene for this division has been identified. The
study with tobacco BY-2 cells showed that Golgi
stacks, mitochondria and plastids sorted them-
selves to distinct subcellular domains during
S128 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
mitosis, but in an apparently cytoskeleton-
independent manner [55,56].
The ER of C. merolae is much simpler than that
of C. caldarium. It forms part of the nuclear envel-
ope with attached ribosomes and did not disappear
during mitosis. This behavior is similar to that of
Saccharyomyces cerevisiae and Schizosacharo-
myces pombe [3]. Yagisawa et al. will report the be-
havior of ER in detail during cell cycle in C.
merolae using antibodies of DD genes. The mother
ER grows and develops during G1, S and G2
phases, divides with cell-nuclear division and is
inherited by the daughter cell-nuclear envelopes
during anaphase. A bridge between the inner and
outer membranes of the nuclear envelope occurs at
the division site of cell nucleus when mother cell
nucleus divides into daughter nuclei (Yagisawa F,
Fujiwara T, Ohnuma M, Nishida K, Imoto Y, Yoshida
Y, Kuroiwa H and Kuroiwa T. unpublished data).
The molecular mechanism of ER inheritance (div-
ision and separation) is unknown.
C. merolae cell has a single Golgi apparatus with
few cisternae in the cytoplasm during the G1 phase
[57], while unicellular green algae and plants
contain more than 200 located around the cell
nucleus and throughout the cytoplasm [47].
Although Noguchi [50,52] and Ueda [51] reported
that the Golgi apparatus divided during the M phase
and the premitotic stage, respectively; however, the
mother Golgi apparatus in C. merolae divided into
daughter Golgi apparatuses in the regions at the
two ends of the cell nucleus during late G1 phase
and the daughters subsequently moved near to the
centrosomes during the G2 phase (Yagisawa F,
Fujiwara T, Ohnuma M, Nishida K, Imoto Y, Yoshida
Y, Kuroiwa H and Kuroiwa T. unpublished data).
After cytokinesis, each daughter cell contained one
Golgi apparatus. Although protein synthesis is
required for division of the Golgi apparatus, its mol-
ecular mechanism being unknown.
When the number of ER and Golgi apparatuses
per cell is small, the behavior of these organelles is
closely connected with the cell nucleus, which acts
as a carrier. When there are a large number of
these organelles, they are distributed through the
entire cytoplasm, as well as close to the cell
nucleus. Interestingly, even when the number of the
organelles is large, their divisions are synchronized
as shown in Micrasterias [51].
Although there must be a molecular mechanism
to control the relationship between these organelle
divisions and the cell nucleus, it is unknown.
Division behavior of lysosomes (vacuoles) and
microbodies ( peroxisomes)
As lysosomes and microbodies function more or
less independently from the synthetic function of
the cell nucleus, the division (inheritance) of these
organelles is not directly related to the division of
cell nuclei but to the division of mitochondria. The
functions of lysosomes and microbodies suggest
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that the origins of these organelles during eukary-
otic evolution may have been later than that of the
ER and the Golgi apparatus. During inheritance
(division, partition) of lysosomes and microbodies,
mitochondria are used as a carrier.
Lysosomes and vacuoles are lytic compartments
and they function as reservoirs for ions and metab-
olites, including pigments, and are crucial for the
processes of detoxification and general cell homeo-
stasis [58]. The vacuoles of algae, plants and yeasts
share some of their basic properties with the lyso-
somes of animal cells [57]. In humans, loss of lysoso-
mal function causes lysosomal storage disorders
such as Fabry disease and GM1 gangliosidosis [59].
Recently, lysosomes have been actively studied as a
central organelle concerned with autophagy. The
main genes for autophagy, apg1-15, have been
actively studied in both yeast and animals [60].
However, all of these genes were not encoded in the
C. merolae genome [9,12]. In mammals, plants and
yeasts, lysosomes are inherited by the daughter cells
[61–63]. In mammalian Madin–Darby canine kidney
(MDCK) cells, video and confocal microscopy ana-
lyses have showed that endosomes and lysosomes
remained intact and separated during mitosis. The
segregation into daughter cells took place by coordi-
nated movements, and the organelles accumulated
in the vicinity of the microtubule organization center
during cytokinesis [61]. In plants, dynamic changes
in vacuoles during mitosis were examined by moni-
toring the tubular structures of the vacuolar mem-
brane (TVM) in living transgenic tobacco BY-2 cells

stably expressing GFP-AtVam3p. TVMs, which were
initially organized from large vacuoles, elongated to
encircle the spindle at metaphase. Subsequently, the
TVMs invaded the equatorial region during anaphase
to telophase, and were divided between the two
daughter cells by the cell plate at cytokinesis.
Furthermore, actin filaments were indispensable for
the development and maintenance of TVMs [62].
However, the molecular mechanisms of lysosome
inheritance were not revealed. During vacuole
inheritance in S. cerevisiae, the vacuole formed ves-
icular–tubular projections known as segregation
structures. The segregation structures originated
from the vacuole membrane and extended to the
daughter bud. This inheritance was based on an
actin cable and myosin-V motor protein Myo2
withVac8 and Vac17 [63,64]. The Myo2-Vac8-Vac17
complex drives the segregation structures of the
vacuole to the bud along the act in cable.
In the C. merolae cell, there are no actin or
myosin filaments. Lysosomes are inherited by
daughter cells by binding to the dividing mitochon-
drion through a small electron-dense bridge
[9,10,65]. No functional actin filaments have been
detected by immunological methods or by staining
with rhodamine-conjugated phalloidin [32]. A single
act in gene was encoded in the C. merolae genome,
but not expressed, while no myosin gene was
present [9]. Therefore, a currently unknown mech-
anism that does not depend on actin filaments must
be involved in vacuole inheritance. Moreover, mam-
malian, plant and yeast cells contain many orga-
nelles whose divisions occur at random, and
organelles with diverse and complicated shapes
[38]. In these cells, the movements of organelles is
not entirely understood.
As described above, C. merolae cell is expected
to provide unique tools for resolving the issue of
lysosome inheritance. Recently, Fujiwara et al.
studied vacuole inheritance ( partition and separ-
ation) during mitosis. Genes involved in this
process were identified by gene expression profiling
[17]. C. merolae contained about three vacuoles that
were equally inherited by the daughter cells by
binding to dividing mitochondria. The binding was
mediated by a novel30-kDa coiled-coil protein
(vacuole inheritance gene 1, VIG1), identified by
microarray analyses and immunological assays
Y. Imoto et al. Cell cycle and organelle division cycles S129
(Fig. 6a). VIG1 appeared on the surface of free
vacuoles in the cytosol, and then tethered the vacu-
oles to the mitochondria by constructing net-like
structures. The vacuoles were released from the
mitochondrion in the daughter cells following VIG1
digestion. Inhibition of VIG1 by cycloheximide or
antisense RNA disturbed the migration of the vacu-
oles and they were unequally inherited by the
daughter cells. VIG1 is essential for vacuole inheri-
tance in C. merolae. Since VIG1 is conserved
among eukaryotes, VIG1 may regulate the inheri-
tance of eukaryote lysosome and vacuoles. The
growth and division of the vacuoles occurred
during G1 and S phases. Separation of the (approxi-
mately) six daughter lysosomes occurred during G2
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and M phases after moving toward and attachment
to the mitochondrion. After completion of MD, the
vacuoles remained attached to the daughter mito-
chondria for more than 1 h before being released
[10,17].
Microbodies ( peroxisomes) are spherules,
0.5–2.0 μm in diameter, and are characterized by
the presence of the enzyme catalase, which pro-
tects the cell from oxidative stress, such as that
caused by peroxide produced following the
exposure of cellular metabolites to high concen-
trations of molecular oxygen. Microbodies play a
role in β-oxidation of fatty acids. They are present
in a wide variety of eukaryotic cells, including
Amoebozoa, Bikonts and Opisthokonts. The
number of microbodies ranges from 1 per cell in
primitive organisms like C. merolae to between 10
and 1000 per cell in Bikonts and Opisthokonts.
Microbody functions are relatively independent
from the cell nucleus they do not associate with
the cell nucleus. Microbodies, like mitochondria
and plastids, are believed to be organelles of bac-
terial origin [66,67]. However, a microbody division
gene of bacterial origin is not yet known in the
eukaryotes. Microbodies are believed to have been
acquired by primitive eukaryotic cells by endosym-
biotic phagocytosis of a prokaryote ( probably an
aerobic Gram-positive bacterium such as Listeria
or Mycoplasma), which has eventually lost its
DNA. This may explain why microbodies behave
more independently from the cell nucleus than the
other single-membrane-bounded organelles. In an
earlier review, we considered that after the
S130 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011

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Fig. 6 Division mechanisms of lysosomes (vacuoles) and microbodies ( peroxisomes). (a) Immunofluorescent and electron
microscopyimages of VIG1 in C. merolae cells. (a) Phase contrast immunofluorescence images of VIG1 (green) and lysosome (red) through
cell cycle. Scale bar, 2 μm (b) Immuno-electron microscopy images of VIG1 in MD. Magnified image of the vacuole region shows binding to
the mitochondrial membrane. vc, vacuole; mt, mitochondria; pt, plastid. Scale bar, 200 nm. (c) Model for vacuole inheritance in C. merolae.
VIG1 accumulates at patch structures on the vacuolar surface in the cytosol. The patch structures have an asymmetric distribution during
S phase. During M phase, the VIG1 patch localizes between the vacuole membrane and mitochondrial surface and tethers the vacuole to the
mitochondria. After cytokinesis, digestion of VIG1 allows the vacuole tore turn to the cytosol. This figure includes photographs that appeared
in Ref. [17] (a–c). (b)Immunofluorescent and electron microscopy images showing segregation of microbodies in C. merolae cells. (a, b)
Phase contrast immunofluorescence images of division of microbodies (green), mitochondria (red) and α-tubulin (blue) at M phase (a) and
cytokinesis (b). Segregating microbodies are in contact with daughter mitochondria (white double arrowheads). (c–e) Electron micrographs
of thin sections of C. merolae cells at M phase (c) and cytokinesis (d). Magnified view of the contact site between a microbody and the
mitochondrion of a cell at the same stage as (d), (e). Electron-dense apparatuses were observed between the microbodies and mitochondria
(black double arrowheads). *or arrowheads, microbodies; cp, chloroplast; mt, mitochondrion. Scale bar, 2 μm (a, b), 500 nm (c, d), 100 nm
(e). (f ) Model for microbody segregation during M phase and cytokinesis in C. merolae. Microbodies bind to centrioles through mitochondria
and electron-dense apparatus. Electron-dense apparatus between the microbody and the mitochondrion play a role in segregating the
microbodies during M phase and cytokinesis, and correspond to the kinetochore of mitotic chromosome division. This figure includes
photographs that appeared in Refs. [21] (c) and [65] (d, e).

bacterial ancestor entered the eukaryote cell, it
lost its DNA and the bacterial cell membrane, and
eventually the bacterial matrix and the interspace
between the outer and inner envelopes became
mixed into the putative pre-microbody [38].
There have been many investigations into the
mechanism of division (inheritance) of microbodies.
A relationship has been reported between microbo-
dies in animal cells and division genes [38].
Generally, microbody divisions in mammalian cells,

trypanosomes, yeasts and algae progress in the
order of elongation, constriction and fission. The
combined efforts of several research groups have
identified 32PEX genes that contribute to the biogen-
esis or maintenance of microbodies. Microbody div-
ision involved the conserved PEX 11 membrane
proteins and, in yeast, was shown to require a
dynamin-like protein. PEX11 and two PEX11-related
proteins were the predominant membrane proteins
of the microbodies of Trypanosoma brucei and it
was concluded that the PEX11 family of proteins
played important roles in determining microbody
membrane structure [68]. Higher level expression of
Pex11pb promotedmicrobody division in mamma-
lian cells [69]. Dynamin-like protein 1 (DLP1), which
is essential for MD, was recently reported to also be
involved in microbody division [70] and was
recruited to microbodies in part by PEX11. Li and
Gould [71] showed that DLP1, the human homolog
of the yeast DNM1 and VPS genes, played an impor-
tant role in microbody division in human cells. In
addition, Fis1p also was found in microbodies. In
conjunction with DLP1, it appears to support the
fission not only of mitochondria, but also of micro-
bodies [70]. Fts1 plays important roles in microbody
division and the maintenance of microbody mor-
phology in mammalian cells, possibly in a concerted
manner together with Pex11pb and DLP1 [72].
However, PEX, dynamin-like proteins, and Fis1p
have not been visualized at the division site of micro-
bodies so that the mechanism of division is still
unclear.
In C. merolae cells, there is a single spherical
microbody which facilitates observation of its
dynamic behavior during cell cycle. Miyagishima
et al. [65] reported that, during the early and middle
G1 phases, the microbody was located in the cyto-
plasm. During late G1 and S phases, the microbody
moved toward its carrier, the mitochondrion, and
attached to it. In the M phase of C. merolae cells,
the mitochondrion and the associated microbody
assumed a characteristic sequence of shapes (in
order: rod, worm, branched, H-shaped, dumb-bell),
and symmetric fission occurred just before cytokin-
esis. The microbody doubled its volume in M phase
and three-dimensional quantitative analysis revealed
that its surface area increased before its volume did
[65]. The microbody was in contact with the
Y. Imoto et al. Cell cycle and organelle division cycles S131
mitochondrion throughout its proliferation cycle,
except in G1 phase cells, and was entwined around
the divisional plane of the mitochondrion during
the MD. Immunocytochemical labeling of catalase
as a marker of matrix proteins of the microbody
revealed that the duplication of catalase occurred
in tandem with the volume increase. The growth
phase of the microbody occurred over about 50 min
during G2 and early prophase. The microbody div-
ision phase occurred over a period of about 130
min from the M phase after completion of MD to
cytokinesis. Thus, microbody was spherical for
about 16 h in G1 phase.
The PEX11 and Fis1p genes were not encoded in
the C. merolae genome and thus these proteins
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were never visualized at the division site of the
microbody. However, Miyagishima et al. [65] ident-
ified an electron-dense apparatus, 30–50 nm in
diameter, between the microbody and the mito-
chondrion, which might play a role in segregating
the microbodies corresponding to the kinetochores
of mitotic chromosomes. Therefore, microbodies
were bound to spindle pole bodies through mito-
chondria [20] (Fig. 6b). Finally, microbodies divided
by binary fission after constriction at their equators.
The composition of putative microbody division
machinery responsible for dividing the microbody
at the division site has not been determined.
Therefore, the mitochondrion might play a role in
separating the daughter microbodies.
These results clearly showed that two daughter
microbodies arose by binary fission of the pre-
existing mother microbody. In animal cells, it is
believed that PEX11 and Fis1 are involved in micro-
body division. However, PEX11 and Fis1 genes for
the microbody division were not encoded in C.
merolae genome. It seems that the genes for MD
induced the microbody division indirectly by means
of the electron dense disc-shaped apparatus.
Cytoskelton and spindle
Cytoskeletal proteins play a major role in the cell
transport system and in division during the mitotic
cycle so that it is important to examine their
dynamics during the cell cycle. Higher eukaryotes
possess complex microtubule systems and a large
number of organelles. In contrast, in C. merolae,
S132 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
two actin genes are not expressed [9,40], the
myosin gene is absent from the genome [9,12], only
five kinesin motor proteins are expressed [9], and
microtubules are only present when they are orga-
nized to form spindles during mitosis [40]. These
unique systems in primitive eukaryotes suggest that
microtubule systems first evolved in association
with the mitotic apparatus and that the cytoskeletal
or transport systems seen in higher eukaryotes
were acquired later. Microtubules are fundamentally
important cytoskeletal elements for nuclear and
organelle division.
C. merolae serves as a model system for the
study of general mechanisms of proliferation of the
cell nucleus and of organelles. Imoto et al. [20]
have characterized the relationships between the
mitotic, mitochondrial, plastid and microbody div-
ision cycles and, using microfluorometry and
cytochemical techniques, have demonstrated the
involvement of microtubules and spindle poles in
these processes. C. merolae cells demonstrated five
discrete stages of microtubule dynamics: (1) the
microtubules disappeared during the G1 phase; (2)
α-tubulin was dispersed within the cytoplasm
without forming microtubules during the S phase;
(3) α-tubulin was assembled into spindle poles
during the G2 phase; (4) polar microtubules were
organized along the mitochondrion during pro-
phase; and (5) mitotic spindles in cell nuclei were
organized during the M phase. Microfluorometry
demonstrated that the location of the intensity peak
corresponding to α-tubulin changed in the following
sequence: spindle poles, mitochondria, spindle
poles and central spindle area. However, the total
fluorescent intensity did not change markedly
throughout the mitotic phases, suggesting that div-
ision and separation of the cell nucleus and mito-
chondrion were mediated by the spindle pole
bodies. Inhibition of microtubule organization
induced inhibitions of cell-nuclear division, mito-
chondrial separation, and division of the single
membrane-bound microbody, implying that, besides
cell-nuclear division, mitochondrial separation and
microbody division were also dependent on micro-
tubules. In the case of centrosomes, the duplication
phase probably occurred over about 2 h during S
phase. The mother centrosomes were separated
into two and α-tubulin was assembled into the
spindle poles for about 1 h during the G2 phase.
Mitochondrial spindles appeared during M phase
( prophase) taking about 200 min. Mitotic spindles
were formed over a period of about 2 h from meta-
phase to the completion of cytokinesis.
Reciprocal relationships among
the organelles
The origin of eukaryotic host cells is still unclear,
but it is now generally accepted that mitochondria
and plastids arose from endosymbiosis of
α-proteobacteria and cyanobacteria-like photosyn-
thetic bacteria, respectively. Many of the genes of
these endosymbionts, including those for DNA
Downloaded from http://jmicro.oxfordjournals.org/ by guest on October 2, 2016
replication and for the maintenance of genomic
integrity, were subsequently lost or transferred to
the nuclear genome by endosymbiotic gene trans-
fer. According to the current dogma, the transfer of
genes of endosymbiotic origin to the cell nucleus
that are required for organelle DNA replication
(ODR) has resulted in loss of the independence of
the cell cycles of the endosymbionts and in their
integration into the eukaryotic control system that
is mediated largely by cyclins and cyclin-dependent
kinase. However, coordination of cell cycle, events
such as DNA replication would have been essential
for establishing the integrity of eukaryotic cells, at
least during the early stages of endosymbiotic
association.
Although there is little evidence for discrete cell-
cycle control of ODR in animal and fungal cells,
studies using algae and flowering plants have shown
that ODR precedes the subsequent cell proliferation
cycles composed of cell nuclear DNA synthesis and
cell division. The advantage of studying cell nuclear
and organelle proliferation cycles in C. merolae is
that cell division can be highly synchronized by
dark–light cycles. Initiation of the cell cycle by illu-
mination in combination with microscopic quantifi-
cation of organelle DNA content revealed that ODR
always occurs before cell nuclear DNA replication
(NDR) [36]. After completion of both ODR and NDR,
division of the plastid, mitochondrion and cell
nucleus occurs sequentially and is followed by cyto-
kinesis. However, the mechanism by which replica-
tion of three genomes with different origins is
coordinated is largely unknown. Kobayashi et al.
 Y. Imoto et al. Cell cycle and organelle division cycles S133

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Fig. 7 Summary of the reciprocal relationships among the double membrane-bounded organelles (cell nucleus, mitochondrion and plastid),
single membrane-bounded organelles (ER, Golgi apparatus, vacuoles/lysosomes and microbody) and non-membrane-bounded organelle
(centrosome). Bold lines indicate cell nucleus–organelle regulations. Division of the mitochondria, plastids and centrosomes are directly
regulated by the cell nucleus through the MD/PD machineries or cytoskeletal proteins such as tubulin. Continuous thick lines indicate
organelle–cell nucleus or organelle–organelle regulations. NDR depends on ODR and is regulated by a tetrapyrrole signal which activates
CDKA in plant cells. Division of the cell nucleus and separation of mitochondria is regulated by centrosomes through mitotic spindles and
mitochondrial spindles. Lysosomes/vacuoles are equally inherited by the daughter cells by interaction with dividing mitochondria mediated
by VIG1. In segregating the microbodies, an electron-dense apparatus, 30–50 nm in diameter, between the microbody and the mitochondrion,
might play an important role. Dashed line shows probable relationships suggested in the present study. Organelle–cell nucleus coupling and
organelle–organelle coupling also seem to have a role in the proliferation of the double-, single-, and non-membrane-bounded organelles.

[73] showed that, in plant cells, NDR was regulated
by a tetrapyrrolesignal, which has been suggested to
be an organelle-to-nucleus retrograde signal. In
synchronized cultures of C. merolae, specific inhi-
bition of A-type cyclin-dependent kinase (CDKA)
prevented NDR but not ODR after onset of the cell
cycle [73]. In contrast, inhibition of ODR by nalidixic
acid also resulted in inhibition of NDR, indicating a
strict dependence of NDR on ODR. The requirement
for ODR to precede NDR was circumvented by
addition of the tetrapyrrole intermediates protopor-
phyrin IX (ProtoIX) or Mg-ProtoIX, both of which
activated CDKA without inducing ODR. This scheme
was also observed in cultured tobacco BY-2 cells,
where inhibition of ODR by nalidixic acid prevented
CDKA activation and NDR, and these inhibitions
were circumvented by Mg-ProtoIX without inducing
ODR. These observations thus show that tetrapyr-
role-mediated organelle–nucleus replicational coup-
ling was an evolutionary conserved process among
plant cells.
Single-membrane-bounded organelles (ER, Golgi
apparatus, vacuoles/lysosomes microbodies) must
originally have had their own division cycles, as
implied by current cytological and morphological
knowledge. Physical attachment and metabolic con-
nection between organelles are now achieved
through interactions between them, such as a mito-
chondrion–vacuole interaction mediated by the
coiled-coil protein VIG1 [17], and a fatty
acidβ-oxidation system which coexists within and
cooperates between mitochondria and microbodies
[74,75]. These physical and metabolic connections
may have developed into the tight reciprocal nets
S134 J O U R N A L O F E L E C T R O N M I C R O S C O P Y , Vol. 60, Supplement 1, 2011
among proliferation cycles in multi-organelle cells
in algae, animals and plants.
Centrosomes are well-known non-mem-brane-
bounded organelles, which regulate the positioning
and transportation of the other double- and
single-membrane-bounded organelles through the
cytoskeleton [76] and the movement of the cell
nucleus by the mitotic spindles. In primitive eukar-
yotes, centrosomes control mitochondrial separation
and microbody division using mitochondrial spindles
[20,40]. Therefore, organelle–cell-nucleus coupling
and organelle–organelle coupling must also be
involved in the proliferation of the double-, single-
and non-membrane-bounded organelles (Fig. 7).
Future directions
Historically, the cell cycle was considered to be rep-
resented by the mitotic cycle consisting of mitosis
and cytokinesis and these processes have been well
elucidated at the molecular level. However, success-
ful cell reproduction requires duplication and segre-
gation (inheritance) of cellular contents, which
includes intracellular organelles as well as the cell-
nuclear genome. In fact, eukaryotic cells contain at
least three double-membrane-bounded organelles
(cell nucleus, mitochondria and plastids), four
single-membrane-bounded organelles (ER, lyso-
somes, Golgi apparatus and microbodies), and the
cytoskeleton containing microtubules and microfila-
ments. These membrane-bounded organelles could
not be formed de novo and must therefore be inher-
ited from parent organelles during cell cycle. We
have proposed that the cell cycle must also include
the division cycles and inheritance of these double-,
single-, and non-membrane-bounded organelles.
Their underlying mechanisms, and the biological
relevance of this process are being clarified, par-
ticularly in the primitive alga C. merolae cells,
which has a minimum of organelles. We have suc-
ceeded in the elucidation of the organelle division
machineries associated with mitochondrial and
plastid division. In future, we expect that the div-
ision cycles of all of these organelles and the reci-
procal relationship among organelles will be
revealed at molecular level and that C. merolae may
provide a glimpse of these fundamental unsolved
problems common to alleukaryotic cells.
Funding
This work was supported by grants from Scientific
Research on Priority Areas (no. 19207004 to T.K.),
the Frontier Project ‘Adaptation and Evolution of
Extremophiles’ from the Ministry of Education,
Culture, Sports, Science and Technology of Japan,
and by the program for the Promotion of Basic
Search Activities for Innovative Biosciences
(PROBRAIN, to T.K.).
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