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60th Anniversary Issue: Biological

The cell cycle, including the mitotic cycle and organelledivision cycles, as revealed by cytological observations

Yuuta Imoto, Yamato Yoshida, Fumi Yagisawa, Haruko Kuroiwaand 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: [email protected], [email protected]

Abstract It is generally believed that the cell cycle consists essentially of themitotic cycle, which involves mitosis and cytokinesis. These processesare 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 thecell-nuclear genome but also intracellular organelles. Eukaryotic cellscontain at least three types of double membrane-bounded organelles (cellnucleus, mitochondria and plastids), four types of single membrane-bounded organelles (endoplasmic reticulum, Golgi apparatus, lysosomesand microbodies) and the cytoskeleton, which comprises tubulin-basedstructures (including microtubules, centrosome and spindle) and actinmicro laments. These membrane-bounded organelles cannot be formedde novo and daughter organelles must be inherited from parent organellesduring cell cycle. Regulation of organelle division and its coordinationwith the progression of the cell cycle involves a sequence of events thatare subjected to precise spatio-temporal control. Considering that thecells 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-boundedorganelles during the cell cycle. Here, we summarize the current cytologi-cal and morphological knowledge of the cell cycle, including the divisioncycles of seven membrane-bounded and some non-membrane-boundedorganelles. 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 byrecent studies.

Keywords cell cycle, organelle-division cycle, mitochondrial-division cycle,chloroplast-division cycle, microbody-division cycle, Golgi apparatus-div-

ision cycleReceived 17 January 2011, accepted 19 April 2011

IntroductionCells reproduce by duplication of their contentsand subsequent division. This cycle of duplicationand division is called the cell cycle, and the events

of the cell cycle are generally understood to corre-spond to the mitotic cycle. Using autoradiography,Howard and Pelc [ 1] discovered that nuclear DNA issynthesized during a stage of interphase designated

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Journal of Electron Microscopy 60(Supplement 1) : S117 – S136 (2011)doi: 10.1093/jmicro/dfr034

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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 identi edin 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 alsocontains 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 organellesas 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 aresubjected to a precise spatio-temporal control. Tounderstand the cell cycle, we need to recognizecoordination between the mitotic cycle and orga-nelle division cycles. Therefore, it is important tosurvey the division cycle of double- andsingle-membrane-bounded organelles and thedynamics of cytoskeleton during the cell cycle.

We have proposed the concept of the ‘mitochon-drial division (MD) cycle ’ analogous to the mitoticcycle [ 4– 6]. In the plasmodium of the slime mold Physarum polycephalum, mitosis and the mitoticcycle are naturally synchronized and, in addition,the mitochondria contain large rod-shaped com- plexes of DNA and proteins, termed nucleoids. Themitotic and MD cycles were precisely examinedusing 3 H-thymidine autoradiography and it wasshown that mitochondrial phases of the divisioncycle, 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, G2and M phases lasted 6, 7 and 1 h, respectively. G1lasted for a very short time. Although the timing of the phases of the mitotic and MD cycles differedmarkedly, the total generation time of the mitoticcycle (14 h) was the same as that of mitochondria (Fig. 1).

The plastid division (PD) cycle was examined intobacco Bright Yellow 2 (BY-2) cells using

cytological techniques. The plastid DNA synthesis phase (pt-S) during the cell cycle of cultured BY-2cells was examined by 3 H-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 alsoobserved in root meristem and cultured BY-2 cellsby immuno uorescence microscopy of Technovitsections using an antibody against 5-bromodeoxyuridine (BrdU) and co- uorescent staining withDAPI and quantitative Southern hybridization [ 8]. Itwas shown that large quantities of both mitochon-drial and plastid DNA were preferentially syn-thesized prior to S-phase. However, in plants,animals and fungi, it has been dif cult 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-phaseand mitotic S-phase has been established.

Partitioning or division (inheritance) of single-mem-brane-bounded organelles occurs in manyorganisms. Additionally, the dynamics of cyto-skeletal proteins and of centrosomes, which playmajor roles in the cell transport system and in celldivision during the mitotic cycle, must be examinedduring 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 eventsand the inner circle depicts cell nuclear events. The duration of each phase is shown in hours. This gure was adapted from Ref. [ 5].

S118 J O UR NA L O F E L E CT RO N M I C RO S CO P Y, Vol. 60, Supplement 1, 2011



orthogonal centrosomes separate. In general, inthese multi-organelle organisms, it has been dif cultto demonstrate each division or partitioning of double-and single-membrane-bounded organellesbecause the cells contain large numbers of theseorganelles and divisions or partitioning occur more

or less randomly (mitochondrial and plastid fusionmay 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-boundedorganelle division cycles. They contain a minimalset 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 fewlysosomes [ 9– 11 ]. Its organelle divisions can be

highly synchronized with the light/dark cycle. Thegenome of C. merolae has been completelysequenced and it is one of the smallest genomes(16.5 Mb), with the lowest number of genes amongfree-living eukaryotes [ 9,12]. Because most of thegenes are present in low copy numbers and lackintrons, they are suitable for identifying functioninggenes by proteome and transcriptome analysis.Moreover, microarray analysis of its geneexpression pro le during organelle division andinheritance was recently reported [ 13].

Besides studies of the mitotic cycle, MD and PDcycles have been examined by genome analysis[9,12], matrix assisted laser desorption ionizationtime of ight mass spectrometry (MALDI-TOF-MS)[14 – 17] and gene targeting [ 15 – 19]. The myosin genewas absent from the genome [ 9,12] and actin geneswere not expressed. Therefore, micro laments andintermediate laments could not be identi edthroughout 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 videoimages of C. merolae during the M phase under phase contrast light microscopy. The images weretaken at 20 min intervals for the rst 220 min after the onset of the chloroplast division, and at 10 minintervals, starting 340 min after the onset of thechloroplast division [ 21]. The temporal sequence of electron microscopic images of the division processof double- and single-membrane-bounded

organelles, and of non-membrane-bounded orga-nelles, during cell cycle could be inferred from theimages of the chloroplasts in living cells.

Here, we review the current cytological and morphological 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). Wediscuss 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 themitotic 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 andthe durations of each phase have been determinedin many organisms, for example, the generationtime 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 phase2.2 h), mitosis (M phase 1.5 h) and the gap betweenM and S phases (G1 phase 1.6 h) [ 22 ,23]. The con-densed mitotic chromosomes are readily visible inalmost all cells of Bikonta, Opisthokonta and Amoebozoa [ 2,3]. In the case of budding yeasts, the16 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-resolutionelectron microautoradiography [ 24 – 27] and the mol-ecular mechanism of chromosome condensation inanimals have been summarized by Hirano [ 28]. Thecell divides by forming a partition and splitting intwo. 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 doesnot break down during M phase. The microtubules

Y. Imoto et al. Cell cycle and organelle division cycles S119



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, onemitochondrion and one chloroplast, simple ER, one Golgi apparatus, one microbody and a few lysosomes. The organelle divisions can behighly synchronized by the light/dark cycle. Chloroplast, mitochondrion and cell nucleus divisions occur in that order. Mitotic chromosomesare 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 thenon-membrane-bounded organelle (centrosome). Double arrowheads indicate the time point of the organelle replication and singlearrowheads indicate the time point of the organelle division. Time shows hours after the second cell cycle.

S120 J O UR NA L O F E L EC T RO N M I C RO S CO P Y, Vol. 60, Supplement 1, 2011



of the mitotic spindle form inside the nucleus andare attached to the spindle pole bodies at its periphery.

Currently, it appears that the cell cycle is composed 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 anER division cycle. Organelle division in C. merolaecan be highly synchronized by a light/dark cycleand, under those conditions, the divisions of Golgiapparatus, chloroplast, mitochondria, microbody,lysosomes, cell-nucleus, ER and nally cytokinesisoccur in that order. Based on studies of slimemolds and higher plants, it has been suggested thatthe cell cycle is composed of a mitotic cycle, a MDcycle 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 epi uores-cence microscopy and uorometry after stainingwith DAPI, using a video-intensi ed microscope photon-counting system (VIMPCS). The mitochon-drial DNA and the chloroplast DNA were syn-thesized during stage I (G1 phase), while thecell-nuclear DNA was duplicated in stage II(S phase). The mitochondrial and chloroplast div-isions began simultaneously in stage II, and chloro-

plast division nished just prior to MD. Themitochondrial and chloroplast-nuclear divisionswere completed in stage IV (M phase). The gener-ation time of this cycle was 28 h. They were able toidentify the duration of each phase of the cellcycles (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 mitoticcycle and chloroplast, mitochondrial, and micro-body division cycles in C. merolae using electronmicroscopy. 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) phasesduring cell cycle, Imoto et al. [20] measured theDNA content of each cell nucleus, mt-nucleus(mt-nucleoid) and pt-nucleus (pt-nucleoid) directlyusing an improved VIMPCS method, after staining

with DAPI. The results were consistent with the previous data. The pt-S phase of the PD cycle andthe mt-S phase of the MD cycle always precededthe S phase of the mitotic cycle by approximately1 h. The division of the plastid was completed rst,followed by the mitochondria, and then, nally, by

the cell nuclei. The durations of each phase of theMD (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 observationsclearly demonstrated that the cell cycle should beunderstood 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 composed of mitotic, MD and PD cycles, which appear to proceed independently of each other. InC. merolae , chloroplasts, mitochondria and cell

nuclei divided in that order, while in the primitivered alga Cyanidium caldarium, the sequence waschloroplasts, cell nuclei and mitochondria [ 32,33].Therefore, there a mechanism that determines theorder of chloroplasts, cell nuclei and MDs must be present. Moreover, in multi-organelle organismssuch 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 observedduring the G1 phase of C. merolae was that the

lower end of the cell-nucleus protruded at thecenter of the mitochondrion, which in turn becamedoughnut-shaped [ 20]. In this cell, the cell-nucleus possessed a single central nucleolus and associatedchromatins, which consisted of a set of three homo-geneous copies of the ribosomal gene and onehistone gene. At this stage, a cytoskeleton and cyto-skeletal proteins (actin, 10 nm laments, tubulin)were absent in cells of C. merolae although cells atG1 phase (10 h) moved actively as amoebae.Therefore, the dynamic changes exhibited by intra-

cellular organelles must have been performedwithout a cytoskeleton. During the S phase, thelower end of cell-nucleus was taken out so that thecell-nucleus containing the nucleolus becamespherical. Tubulin molecules were synthesized asmonomers and they diffused throughout the entirecytoplasm for about 1.5 h during the S phase.Probably, the single mother centrosome core(without microtubules) was formed in the




cytoplasm near the peripheral end of the mitochon-drion during early S phase and it was replicatedand divided into two daughter centrosome coresduring late S phase (unpublished data). For 1 hduring the G2 phase, the cell nucleus becamefootball-shaped. Microtubules were focused on two

daughter centrosome cores and formed centro-somes. For 2 h during the early M phase, thenucleus changed from a football-like structure intoa Napoleon hat-like con guration (inverted cup) of which the two ends were associated with the cen-trosomes. Several microtubules extended from thecentrosomes and were positioned on the mitochon-drion to form a mitochondrial spindle. The nucleo-lus began to move from the central region of thecell nucleus to the upper region. For 2 h, during themiddle M phase, the cell nucleus became peanut-

shaped and homogenous. During metaphase, rod-shaped mitotic chromosomes were never observedalthough condensins (multi-subunit proteincomplex that play a central role in mitotic chromo-some assembly and segregation) [ 28] were presentand a mitotic nuclear spindle was formed betweendaughter centrosomes. Consequently, chromosomecondensation did not occur in C. merolae althoughCENH3-containing kinetochores reconstituted intotwo separate dot and co-localized with centrosomesduring 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 dividedinto daughter cell nuclei by the activity of mitoticspindle. The central spindle then pushed the daugh-ter cell nuclei apart from the daughter cells. Thedaughter nuclei then changed back from being cup-shaped to spherical and cytokinesis occurred in theabsence of an actin cytoskeleton [ 9,32]. After themitotic division, the cell-nucleus with its centralnucleolus resumed a spherical shape, suggesting

that a part of cell nucleus did not protrude intomitochondrion.

Aspects of C. merolae cell division differ fromthat observed in other cells and are summarized asfollows: (a) Tubulin synthesis is con ned 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) Cytokinesisoccurs in the absence of a contractile rings of actin.In preliminary experiments, EF1 α has been locatedin the contractile ring region, and it is likely thatthis protein controls cytokinesis in many eukaryoticcells (Imoto Y, Nishida K, Yagisawa F, Yoshida M,

Yoshida Y, Ohnuma M, Fujiwara T, Kuroiwa H andKuroiwa T. unpublished data) (Fig. 3b).

Mitochondrial behavior

As described above, the concept of the organelledivision cycle has been evoked by the MD cycle of P. polycephalum . Mitochondria have their ownDNA which is organized using basic proteins toform organelle nuclei (nucleoids). Organelle nucleiare universal in eukaryotes and the mechanism of

condensation of mitochondrial nuclei has beensummarized [ 6,35,36]. Mitochondrial nuclei are syn-thesized during a speci c 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: divisionof the mitochondrial nuclei, which is followed bydivision of the matrix (the so-called MD or mito-chondriokinesis). The mechanism of mitochondrialnuclear division has been summarized by Kuroiwa et al. [35].

Mitochondriokinesis occurs after mitochondrialnuclear division and in this respect it resembles bac-terial cytokinesis. We have described the ne struc-ture and dynamics of the MD ring which provide themorphological bases for mitochondriokinesis[37,38]. Electron microscopic studies rst identi eda small electron-dense ring at the constriction sitesof dividing mitochondria in P. polycephalum and,subsequently, similar structures have been observedin Nitella exilis [6], Nannochloropsis oculata [39]and higher plants [ 38]. However, in multi-organelle

organisms such as animal and plants, it was dif cultto examine the timing of each phase of the MD cycleand of the MD ring because their cells have manyorganelles which divide asynchronously, and thering 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




time course has been deduced from uorescenceimages using the VIMPCS system [ 20], and fromliving cells [ 21]. The morphological changes of theorganelles were as follows. For 9 h during early andmiddle 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 themitochondrion became at again, the nucleusbecame 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 elongateddisc-shaped structure and the microbody attachedto the peripheral end of mitochondrion. For 1 hduring 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 theend to the central region of mitochondrion andattached to the outer ring of the MD machinery.The centrosomes of the mitochondrial spindle wereformed at the each end of mitochondrion. For 2 hduring prophase (late mt-G2), the microbody beganelongating along the MD machinery of the cup-shaped mitochondrion and surrounded the outer ring of the MD machinery. The MD machinery thenbegan 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 composed 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 dividedinto two daughter mitochondria. For 2 h duringmetaphase (mt-M phase), the dumbbell-shapedmother microbody divided and separated intospherical daughter microbodies by the activities of

Fig. 3 (a) Immuno uorescent micrographs showing the cell nucleus in C. merolae during G2 and M phase. (a – d) Phase contrastimmuno uorescence images of DAPI (blue), CENH3 (green) indicating centromere and α-tubulin (red) in M phase cell. DAPI-stained cellnucleus (white arrowheads) shows that chromosome condensation did not occur in C. merolae . (e – h) Immuno uorescence 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 gure includes photographs that appeared in Refs [ 20] (a – d) and [ 34] (e – h).(b) Immuno uorescent micrographs showing the early cytokinesis in C. merolae . (a – d) Phase contrast immuno uorescence images of DAPI(blue), EF1 α (green) indicating and α -tubulin (red) in early cytokinesis cell. The signals of EF1 α are detected in the contractile ring region(white double arrow heads).




electron-dense patches (50 nm in diameter) at eachside of the microbody, and microtubules of themitochondrial spindle. During anaphase (mt-M phase), each microbody detached from the daugh-ter mitochondria and mitosis and cytokinesis thenoccurred [ 37,40 – 43]. Recent interesting progress in

the eld is that MD machineries have now been iso-lated from C. merolae cells [ 16]. A novel ZapA-likebacterial protein, ZED, has provided new insightsinto MD and the birth of eukaryotic cells.

Bacterial cell division systems that include FtsZhave been found throughout prokaryotes. Mitochon-dria arose from an endosymbiotic α-proteobacterialancestor 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 identi ed in Escherichia coli mutants collected in the late 1960s.Several proteins involved in bacterial protein recruit-ment to the division site in an FtsZ-dependentmanner had been identi ed, 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 cellcycle, in a manner similar to bacterial division.However, mitochondria have persisted withineukaryotic cells for a long period — approximately 1 –

2 billion years. Since the organelle genome sizes,even in lower eukaryotes, are less than 10% of thesize of the genomes found in free-living bacteria, it isthought that following endosymbiosis, initially about90% of the endosymbiotic bacterial genes were trans-ferred to the host cell nuclear genome, or lostentirely. As a consequence, only one FtsZ gene

remains as the mitochondrial dividing gene. Yoshida et al. [16] have now isolated the MD machinery andidenti ed a protein ZED that constricts the basalstructure of the MD machinery with FtsZ (Fig. 4).ZED contains a mitochondrial transit signal and twocoiled-coil regions and has a partial homology withthe bacterial division protein ZapA. In cytologicalstudies, 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 formthe basic structure of the MD machinery and wasrequired for the MD. Bacterial ZapA and mitochon-drial ZED were also functionally very similar, whichimplies that the bacterial cell division system was

incorporated into the MD machinery during evol-ution with subsequent loss of most of the bacterialdividing genes.

Plastid (chloroplast)behavior

The division of plastids (chloroplasts) is universally present in Biokonta. As mitochondria, plastids 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 theevolution of eukaryotes [ 44]. Dynamic changes of plastid nuclei have been examined during the lifecycle of algae and higher plants and the mechanismof the condensation of plastid nuclei has beenstudied [ 45]. Plastid DNA (nuclei) is synthesizedduring a speci c 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 andthe pt-G1 phase. During the pt-M phase, the divisioncan be separated into two main events: division of

the plastid nuclei and division of the stroma (theso-called plastid division or plastidkinesis). Themechanism of plastid nuclear division is summar-ized below [ 45].

Plastid kinesis occurs after plastid nuclear div-ision and is similar to bacterial cytokinesis, basedon the evidence of the FtsZ protein which isinvolved in plastid division in Arabidopsis thaliana [46,47]. The ne structure and dynamics of the plastid division ring (PD ring) provide the morpho-logical bases of plastid division [ 37 ,38]. Electron

microscopic studies rst identi ed a smallelectron-dense ring at the constriction sites of divid-ing plastids in C. caldarium and subsequent studieshave shown that similar structures are universally present among the Bikonta [ 39]. However, in multi-organelle algae and plants, it is dif cult to examinethe timing of each phase of the plastid divisioncycle because their organelles divide asynchro-nously and the ring is too small to be studied.




C. merolae was also employed to study the morphological changes during plastid division, and toestimate its time course, using uorescence imagingand 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 plastidnucleus became enlarged and, for about 1.5 h duringlate G1 (pt-S), it synthesized DNA. Over 2 h during S phase (pt-G2), the plastid enlarged further andbecame peanut-shaped. The chloroplast nucleusthen elongated at one end and attached itself to theupper wall at one end of the chloroplast. Over a

period of 1 h during G2 phase (pt-M), the plastidnucleus 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 components of the plastid division machinery (FtsZ ringand PD ring) were formed rst, followed by theouter 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

Fig. 4 Immuno uorescent and electron microscope images showing ZED rings of mitochondria in C. merolae cells. (a – e) Phase contrastimmuno uorescence images of ZED (green) and mitochondrial matrix (red) through MD. (f – h) Phase contrast immuno uorescence imagesshow 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. ZEDinteract with FtsZ1 to constitute the basal structure of MD machinery. This gure includes photographs that appeared in reference [ 16] (a – i), (k).




metaphase. The plastid division machinery associ-ated with the daughter plastids was released and

nally broken down. MD, microbody division,mitosis and cytokinesis then follow in that order.The mechanism of chloroplast division by the PDmachinery has been reviewed by Kuroiwa et al.

[38,44].Recently, our understanding of the process of

plastid division machinery has been advanced byisolation of the PD machinery and by identi cationof proteins and other components using MALDI –

TOF – MS [14 ,15 ]. During chloroplast division, theFtsZ ring (localized in the stroma), the PD ring(in the cytoplasm) and the dynamin ring (in thecytoplasm) were formed in that order. The PD ringis the main structural component of the PDmachinery and was observed to be a bundle of ne

laments 5–

7 nm in diameter. Two types of guanosinetriphosphatase, a bacterially derivedFtsZ and a eukaryote-speci c dynamin, have beencharacterized as PD-machinery-associated pro-teins. The isolation and analysis of the PD machin-ery have been achieved and the completesequencing of the genome has enabled proteomicanalysis. To detect the components of the PD ring,Yoshida et al. [15 ,16 ] digested isolated PD machi-neries with various proteases but were unable tocompletely decompose them. Therefore, they

searched for hypothetical components that mightconstitute the PD ring. Electron-dense deposits(indicating carbohydrates) appeared on the PDring after staining with periodic acid – horseradish peroxidase (PA-HRP). This suggested that the PDring might have a saccharic architecture. Tocon rm this, we isolated PD machineries fromdividing cells of C. merolae . Proteomic analysisidenti ed a protein, PDR1, a glycosyltransferasewhich was present in the PD ring and is widelyconserved from red alga to land plants (Fig. 5).

Electron microscopy showed that PDR1 forms a ring with carbohydrates at the chloroplast-divisionsite. Fluorometric saccharide ingredient analysisof puri ed PD ring laments showed that onlyglucose was included, and down-regulation of PDR1 impaired chloroplast division. Thus, thechloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan laments. Asthe PDR-1 gene is universal in Bikonta [ 15], i t is

clearly a key to solving the molecular mechanismof plastid division.

Division cycles and inheritance of single-membrane-bounded organelles(ER, Golgi apparatus, lysosomes andmicrobodies)The inheritance of single-membrane-bounded, andDNA-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.

Division cycle and inheritance of ER and Golgiapparatus 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 cellnucleus and moves to the ER for translation.Proteins are transported from ER to Golgi apparatus,which processes and packages both proteins andlipids. Subsequently, these processes are aided bythe 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 bythe inheritance of the ER and Golgi apparatus inassociation with the mitotic spindle. By contrast, themitochondria are required as the dividing carrier of microbodies and lysosomes which are carried on themitochondrial spindle [ 20]. Therefore, we reviewthat the inheritances of ER and Golgi apparatus arein relation to the cell nucleus, while the inheritancesof microbodies and lysosomes are in relation tomitochondria. Although single-membrane-boundedorganelles 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, anda phase of separation and release from the carrier. As in many organisms, the cells contains numeroussingle-membrane-bounded organelles, it is dif cultto identify each of these phases. By contrast, in cellsof C. merolae cell, which contain a minimum of




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 thecell nucleus. Sheahan et al. [48] reported thatduring 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 theinheritance of the ER is unclear.

By contrast, the division and inheritance of theGolgi apparatus (dictyosome) was cytologicallyexamined 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 apparatusdivision cycles. In the alga Chlorococcum infusio- num , the Golgi apparatus was localized close to thecell nucleus. It was not duplicated during cell wall

formation, but during the binary division of the cell

nucleus [ 50]. The numerous Golgi apparatuses inthe large unicellular green algae Micrasterias sp .were located not only around cell nuclei, but alsothroughout the cytoplasm [ 51]. During the mitotic phase, their numbers increased from about 70 to150 in Micrasterias pinnati da , and from 200 to400 in M. crux-melitensis . These additional Golgiapparatuses, which were distributed throughout theentire cytoplasm in M. pinnati da [52] andClosterium ehrenbergii [53], appeared synchro-nously at the premitotic stage. Noguchi [ 50] con-

cluded that Golgi apparatuses in plant and animalcells divided during the M phase. The mother Golgi apparatus divided at the center from the cisside to the trans side to form daughter Golgiapparatuses [ 54]. However, no special structure or gene for this division has been identi ed. Thestudy with tobacco BY-2 cells showed that Golgistacks, mitochondria and plastids sorted them-selves to distinct subcellular domains during

Fig. 5 Immuno uorescent and electron microscopyimages of PDR1 in C. merolae cells. (a – f) Phase contrast immuno uorescence images of PDR1 (green) and auto uorescence of plastids (red) through plastid division. (g) Electron microscopy using the PA-HRP method in a cell.The PD machinery is stained black. Magni ed image shows spectral colors correspondingto the intensity of staining signals of the regionaround the PD ring (white arrowheads) (see color scales). cp, chloroplast. Scalebar, 2 μm. (h) Immuno-electron microscopy images of PDR1in chloroplast division. Magni ed 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 thechloroplast division site and synthesize F-PDRs (PD ring laments) (black) from UDP-glucose (step 2a). Other PDR1 proteins bind toelongated F-PDRs and synthesize more F-PDRs (step 2b and step 3). This gure includes photographs that appeared in Ref. [ 15].




mitosis, but in an apparently cytoskeleton-independent manner [ 55,56].

The ER of C. merolae is much simpler than thatof 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 isinherited by the daughter cell-nuclear envelopesduring anaphase. A bridge between the inner andouter membranes of the nuclear envelope occurs atthe division site of cell nucleus when mother cellnucleus 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 withfew cisternae in the cytoplasm during the G1 phase[57], while unicellular green algae and plantscontain more than 200 located around the cellnucleus and throughout the cytoplasm [ 47]. Although Noguchi [ 50,52] and Ueda [ 51] reportedthat the Golgi apparatus divided during the M phase

and the premitotic stage, respectively; however, themother Golgi apparatus in C. merolae divided intodaughter Golgi apparatuses in the regions at thetwo ends of the cell nucleus during late G1 phaseand the daughters subsequently moved near to thecentrosomes 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 oneGolgi apparatus. Although protein synthesis isrequired 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 isclosely connected with the cell nucleus, which actsas a carrier. When there are a large number of these organelles, they are distributed through theentire cytoplasm, as well as close to the cellnucleus. Interestingly, even when the number of the

organelles is large, their divisions are synchronizedas shown in Micrasterias [51].

Although there must be a molecular mechanismto control the relationship between these organelledivisions and the cell nucleus, it is unknown.

Division behavior of lysosomes (vacuoles) andmicrobodies (peroxisomes)

As lysosomes and microbodies function more or less independently from the synthetic function of the cell nucleus, the division (inheritance) of theseorganelles is not directly related to the division of cell nuclei but to the division of mitochondria. Thefunctions of lysosomes and microbodies suggestthat 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 compartmentsand they function as reservoirs for ions and metab-olites, including pigments, and are crucial for the processes of detoxi cation and general cell homeo-stasis [ 58]. The vacuoles of algae, plants and yeastsshare 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. Themain genes for autophagy, apg1-15 , have beenactively studied in both yeast and animals [ 60].However, all of these genes were not encoded in theC. 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. Thesegregation into daughter cells took place by coordi-nated movements, and the organelles accumulatedin the vicinity of the microtubule organization center during cytokinesis [ 61]. In plants, dynamic changesin 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 wereinitially organized from large vacuoles, elongated toencircle the spindle at metaphase. Subsequently, theTVMs invaded the equatorial region during anaphaseto telophase, and were divided between the twodaughter cells by the cell plate at cytokinesis.

Furthermore, actin laments were indispensable for the development and maintenance of TVMs [ 62].However, the molecular mechanisms of lysosomeinheritance were not revealed. During vacuoleinheritance in S. cerevisiae , the vacuole formed ves-icular – tubular projections known as segregationstructures. The segregation structures originatedfrom the vacuole membrane and extended to thedaughter bud. This inheritance was based on anactin cable and myosin-V motor protein Myo2withVac8 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 laments. Lysosomes are inherited bydaughter cells by binding to the dividing mitochon-drion through a small electron-dense bridge[9,10,65]. No functional actin laments have beendetected by immunological methods or by stainingwith rhodamine-conjugated phalloidin [ 32]. A singleact 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 laments mustbe involved in vacuole inheritance. Moreover, mam-malian, plant and yeast cells contain many orga-nelles whose divisions occur at random, andorganelles with diverse and complicated shapes[38]. In these cells, the movements of organelles isnot entirely understood.

As described above, C. merolae cell is expectedto 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 identi ed by gene expression pro ling[17]. C. merolae contained about three vacuoles thatwere equally inherited by the daughter cells bybinding to dividing mitochondria. The binding wasmediated by a novel30-kDa coiled-coil protein(vacuole inheritance gene 1, VIG1), identi ed bymicroarray analyses and immunological assays

(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-likestructures. The vacuoles were released from themitochondrion in the daughter cells following VIG1digestion. Inhibition of VIG1 by cycloheximide or

antisense RNA disturbed the migration of the vacu-oles and they were unequally inherited by thedaughter cells. VIG1 is essential for vacuole inheri-tance in C. merolae . Since VIG1 is conservedamong eukaryotes, VIG1 may regulate the inheri-tance of eukaryote lysosome and vacuoles. Thegrowth and division of the vacuoles occurredduring G1 and S phases. Separation of the (approxi-mately) six daughter lysosomes occurred during G2and M phases after moving toward and attachmentto 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 bythe presence of the enzyme catalase, which pro-tects the cell from oxidative stress, such as thatcaused by peroxide produced following theexposure 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. Thenumber of microbodies ranges from 1 per cell in primitive organisms like C. merolae to between 10and 1000 per cell in Bikonts and Opisthokonts.Microbody functions are relatively independentfrom the cell nucleus they do not associate withthe cell nucleus. Microbodies, like mitochondria and plastids, are believed to be organelles of bac-terial origin [ 66,67]. However, a microbody divisiongene of bacterial origin is not yet known in the

eukaryotes. Microbodies are believed to have beenacquired by primitive eukaryotic cells by endosym-biotic phagocytosis of a prokaryote (probably anaerobic Gram-positive bacterium such as Listeriaor Mycoplasma ), which has eventually lost itsDNA. This may explain why microbodies behavemore independently from the cell nucleus than theother single-membrane-bounded organelles. In anearlier review, we considered that after the




bacterial ancestor entered the eukaryote cell, itlost its DNA and the bacterial cell membrane, andeventually the bacterial matrix and the interspacebetween the outer and inner envelopes becamemixed into the putative pre-microbody [ 38].

There have been many investigations into themechanism 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,

Fig. 6 Division mechanisms of lysosomes (vacuoles) and microbodies (peroxisomes). (a) Immuno uorescent and electronmicroscopyimages of VIG1 in C. merolae cells. (a) Phase contrast immuno uorescence images of VIG1 (green) and lysosome (red) throughcell cycle. Scale bar, 2 μm (b) Immuno-electron microscopy images of VIG1 in MD. Magni ed image of the vacuole region shows binding tothe 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 duringS phase. During M phase, the VIG1 patch localizes between the vacuole membrane and mitochondrial surface and tethers the vacuole to themitochondria. After cytokinesis, digestion of VIG1 allows the vacuole tore turn to the cytosol. This gure includes photographs that appearedin Ref. [ 17] (a – c). (b)Immuno uorescent and electron microscopy images showing segregation of microbodies in C. merolae cells. (a, b)Phase contrast immuno uorescence images of division of microbodies (green), mitochondria (red) and α-tubulin (blue) at M phase (a) andcytokinesis (b). Segregating microbodies are in contact with daughter mitochondria (white double arrowheads). (c – e) Electron micrographsof thin sections of C. merolae cells at M phase (c) and cytokinesis (d). Magni ed view of the contact site between a microbody and themitochondrion 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 themicrobodies during M phase and cytokinesis, and correspond to the kinetochore of mitotic chromosome division. This gure includes photographs that appeared in Refs. [ 21] (c) and [ 65] (d, e).

S130 J O UR NA L O F E L E CT RO N M I C RO S CO P Y, Vol. 60, Supplement 1, 2011



trypanosomes, yeasts and algae progress in theorder of elongation, constriction and ssion. Thecombined efforts of several research groups haveidenti ed 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 proteinsof the microbodies of Trypanosoma brucei and itwas concluded that the PEX11 family of proteins played important roles in determining microbodymembrane structure [ 68]. Higher level expression of Pex11pb promotedmicrobody division in mamma-lian cells [ 69]. Dynamin-like protein 1 (DLP1), whichis essential for MD, was recently reported to also beinvolved in microbody division [ 70] and was

recruited to microbodies in part by PEX11. Li andGould [ 71] showed that DLP1 , the human homologof the yeast DNM1 and VPS genes, played an impor-tant role in microbody division in human cells. Inaddition, Fis1p also was found in microbodies. Inconjunction with DLP1, it appears to support the

ssion not only of mitochondria, but also of micro-bodies [ 70]. Fts1 plays important roles in microbodydivision and the maintenance of microbody mor- phology in mammalian cells, possibly in a concertedmanner together with Pex11pb and DLP1 [ 72].

However, PEX, dynamin-like proteins, and Fis1phave not been visualized at the division site of micro-bodies so that the mechanism of division is stillunclear.

In C. merolae cells, there is a single sphericalmicrobody which facilitates observation of itsdynamic behavior during cell cycle. Miyagishima et al. [65] reported that, during the early and middleG1 phases, the microbody was located in the cytoplasm. During late G1 and S phases, the microbodymoved toward its carrier, the mitochondrion, and

attached to it. In the M phase of C. merolae cells,the mitochondrion and the associated microbodyassumed a characteristic sequence of shapes (inorder: rod, worm, branched, H-shaped, dumb-bell),and symmetric ssion occurred just before cytokin-esis. The microbody doubled its volume in M phaseand three-dimensional quantitative analysis revealedthat its surface area increased before its volume did[65]. The microbody was in contact with the

mitochondrion throughout its proliferation cycle,except in G1 phase cells, and was entwined aroundthe divisional plane of the mitochondrion duringthe MD. Immunocytochemical labeling of catalaseas a marker of matrix proteins of the microbodyrevealed that the duplication of catalase occurred

in tandem with the volume increase. The growth phase of the microbody occurred over about 50 minduring G2 and early prophase. The microbody div-ision phase occurred over a period of about 130min from the M phase after completion of MD tocytokinesis. Thus, microbody was spherical for about 16 h in G1 phase.

The PEX11 and Fis1p genes were not encoded inthe C. merolae genome and thus these proteinswere never visualized at the division site of themicrobody. However, Miyagishima et al. [65] ident-

i ed an electron-dense apparatus, 30–

50 nm indiameter, between the microbody and the mito-chondrion, which might play a role in segregatingthe microbodies corresponding to the kinetochoresof mitotic chromosomes. Therefore, microbodieswere bound to spindle pole bodies through mito-chondria [ 20] (Fig. 6b). Finally, microbodies dividedby binary ssion after constriction at their equators.The composition of putative microbody divisionmachinery responsible for dividing the microbodyat the division site has not been determined.

Therefore, the mitochondrion might play a role inseparating the daughter microbodies.

These results clearly showed that two daughter microbodies arose by binary ssion of the pre-existing mother microbody. In animal cells, it isbelieved 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 MDinduced the microbody division indirectly by meansof the electron dense disc-shaped apparatus.

Cytoskelton and spindleCytoskeletal proteins play a major role in the celltransport system and in division during the mitoticcycle so that it is important to examine their dynamics during the cell cycle. Higher eukaryotes possess complex microtubule systems and a largenumber of organelles. In contrast, in C. merolae,




two actin genes are not expressed [ 9,40], themyosin gene is absent from the genome [ 9,12], only ve kinesin motor proteins are expressed [ 9], and

microtubules are only present when they are orga-nized to form spindles during mitosis [ 40]. Theseunique systems in primitive eukaryotes suggest that

microtubule systems rst evolved in associationwith the mitotic apparatus and that the cytoskeletalor transport systems seen in higher eukaryoteswere acquired later. Microtubules are fundamentallyimportant cytoskeletal elements for nuclear andorganelle division.

C. merolae serves as a model system for thestudy of general mechanisms of proliferation of thecell nucleus and of organelles. Imoto et al. [20]have characterized the relationships between themitotic, mitochondrial, plastid and microbody div-

ision cycles and, using micro uorometry andcytochemical techniques, have demonstrated theinvolvement of microtubules and spindle poles inthese processes. C. merolae cells demonstrated vediscrete stages of microtubule dynamics: (1) themicrotubules disappeared during the G1 phase; (2)α -tubulin was dispersed within the cytoplasmwithout forming microtubules during the S phase;(3) α-tubulin was assembled into spindle polesduring the G2 phase; (4) polar microtubules wereorganized along the mitochondrion during pro-

phase; and (5) mitotic spindles in cell nuclei wereorganized during the M phase. Micro uorometrydemonstrated that the location of the intensity peakcorresponding to α -tubulin changed in the followingsequence: spindle poles, mitochondria, spindle poles and central spindle area. However, the total

uorescent intensity did not change markedlythroughout the mitotic phases, suggesting that div-ision and separation of the cell nucleus and mito-chondrion were mediated by the spindle polebodies. Inhibition of microtubule organization

induced inhibitions of cell-nuclear division, mito-chondrial separation, and division of the singlemembrane-bound microbody, implying that, besidescell-nuclear division, mitochondrial separation andmicrobody 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 separatedinto 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 spindleswere formed over a period of about 2 h from metaphase to the completion of cytokinesis.

Reciprocal relationships amongthe organellesThe 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 replication and for the maintenance of genomicintegrity, 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 nucleusthat 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 thatis mediated largely by cyclins and cyclin-dependentkinase. However, coordination of cell cycle, eventssuch as DNA replication would have been essentialfor establishing the integrity of eukaryotic cells, at

least during the early stages of endosymbioticassociation.

Although there is little evidence for discrete cell-cycle control of ODR in animal and fungal cells,studies using algae and owering plants have shownthat ODR precedes the subsequent cell proliferationcycles composed of cell nuclear DNA synthesis andcell division. The advantage of studying cell nuclear and organelle proliferation cycles in C. merolae isthat cell division can be highly synchronized bydark – light cycles. Initiation of the cell cycle by illu-

mination in combination with microscopic quanti -cation of organelle DNA content revealed that ODRalways occurs before cell nuclear DNA replication(NDR) [ 36]. After completion of both ODR and NDR,division of the plastid, mitochondrion and cellnucleus occurs sequentially and is followed by cyto-kinesis. However, the mechanism by which replica-tion of three genomes with different origins iscoordinated is largely unknown. Kobayashi et al.




[73] showed that, in plant cells, NDR was regulatedby a tetrapyrrolesignal, which has been suggested tobe an organelle-to-nucleus retrograde signal. Insynchronized cultures of C. merolae , speci c inhi-bition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cellcycle [ 73]. In contrast, inhibition of ODR by nalidixicacid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirementfor ODR to precede NDR was circumvented byaddition of the tetrapyrrole intermediates protopor- phyrin IX (ProtoIX) or Mg-ProtoIX, both of whichactivated CDKA without inducing ODR. This schemewas also observed in cultured tobacco BY-2 cells,where inhibition of ODR by nalidixic acid preventedCDKA activation and NDR, and these inhibitionswere 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, Golgiapparatus, vacuoles/lysosomes microbodies) mustoriginally have had their own division cycles, asimplied by current cytological and morphologicalknowledge. Physical attachment and metabolic con-nection between organelles are now achievedthrough interactions between them, such as a mito-chondrion – vacuole interaction mediated by thecoiled-coil protein VIG1 [ 17], and a fattyacid β -oxidation system which coexists within andcooperates between mitochondria and microbodies[74,75]. These physical and metabolic connectionsmay have developed into the tight reciprocal nets

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 directlyregulated by the cell nucleus through the MD/PD machineries or cytoskeletal proteins such as tubulin. Continuous thick lines indicateorganelle – cell nucleus or organelle – organelle regulations. NDR depends on ODR and is regulated by a tetrapyrrole signal which activatesCDKA in plant cells. Division of the cell nucleus and separation of mitochondria is regulated by centrosomes through mitotic spindles andmitochondrial spindles. Lysosomes/vacuoles are equally inherited by the daughter cells by interaction with dividing mitochondria mediatedby 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 andorganelle – organelle coupling also seem to have a role in the proliferation of the double-, single-, and non-membrane-bounded organelles.




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among proliferation cycles in multi-organelle cellsin algae, animals and plants.

Centrosomes are well-known non-mem-brane-bounded organelles, which regulate the positioningand transportation of the other double- andsingle-membrane-bounded organelles through the

cytoskeleton [ 76] and the movement of the cellnucleus by the mitotic spindles. In primitive eukaryotes, centrosomes control mitochondrial separationand microbody division using mitochondrial spindles[20,40]. Therefore, organelle – cell-nucleus couplingand organelle – organelle coupling must also beinvolved 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 mitosisand cytokinesis and these processes have been wellelucidated at the molecular level. However, success-ful cell reproduction requires duplication and segre-gation (inheritance) of cellular contents, whichincludes intracellular organelles as well as the cell-nuclear genome. In fact, eukaryotic cells contain atleast three double-membrane-bounded organelles(cell nucleus, mitochondria and plastids), four single-membrane-bounded organelles (ER, lyso-

somes, Golgi apparatus and microbodies), and thecytoskeleton containing microtubules and micro la-ments. These membrane-bounded organelles couldnot be formed de novo and must therefore be inher-ited from parent organelles during cell cycle. Wehave proposed that the cell cycle must also includethe division cycles and inheritance of these double-,single-, and non-membrane-bounded organelles.Their underlying mechanisms, and the biologicalrelevance of this process are being clari ed, 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 divisionmachineries associated with mitochondrial and plastid division. In future, we expect that the div-ision cycles of all of these organelles and the reciprocal relationship among organelles will berevealed at molecular level and that C. merolae may provide a glimpse of these fundamental unsolved problems common to alleukaryotic cells.

FundingThis work was supported by grants from Scienti cResearch 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 BasicSearch Activities for Innovative Biosciences(PROBRAIN, to T.K.).

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