SHORT COMMUNICATION

Golgi inheritance in the primitive red alga,Cyanidioschyzon merolae

Fumi Yagisawa & Takayuki Fujiwara & Mio Ohnuma &

Haruko Kuroiwa & Keiji Nishida & Yuuta Imoto &

Yamato Yoshida & Tsuneyoshi Kuroiwa

Received: 1 August 2012 /Accepted: 28 October 2012# Springer-Verlag Wien 2012

Abstract The Golgi body has important roles in modi-fying, sorting, and transport of proteins and lipids.Eukaryotic cells have evolved in various ways to inheritthe Golgi body from mother to daughter cells, whichallows the cells to function properly immediately aftermitosis. Here we used Cyanidioschyzon merolae, one of themost suitable systems for studies of organelle dynamics, toinvestigate the inheritance of the Golgi. Two proteins, Sed5and Got1, were used as Golgi markers. Using immunofluo-rescence microscopy, we demonstrated that C. merolae con-tains one to two Golgi bodies per cell. The Golgi body waslocalized to the perinuclear region during the G1 and S phasesand next to the spindle poles in a microtubule-dependentmanner duringM phase. It was inherited together with spindlepoles upon cytokinesis. These observations suggested thatGolgi inheritance is dependent on microtubules inC.merolae.

Keywords Golgi inheritance . Cyanidioschyzon .

Immunofluorescence microscopy . Sed5 . Microtubule

Introduction

The Golgi body harbors a series of enzymes involved inprotein modification and lipid synthesis. During mitosis, theGolgi body is inherited from mother cells to daughter cellsin a range of eukaryotes, which allows cells to functionproperly immediately after cell division (Lowe and Barr2007). The ways in which the Golgi is inherited appear tovary, reflecting the diverse structures, copy numbers percell, and mechanisms for biogenesis of this organelle. Twomodels have been proposed in mammalian cells, where theGolgi bodies are present as a single perinuclear Golgi ribbonduring interphase. In one model, the Golgi ribbon breaksdown into vesicles that disperse throughout the cytoplasm atthe onset of mitosis. Subsequently, the vesicles develop intothe new Golgi body in daughter cells (Warren and Wickner1996; Presico et al. 2009). Recent studies have shown thatthe breakdown of the Golgi ribbon starts from the fragmen-tation of the non-compact zones of the Golgi ribbon in G2phase, which results in the formation of isolated Golgibodies or small groups of Golgi bodies (Presico et al.2009). In another model, the Golgi body is absorbed intoand then re-emerges from the endoplasmic reticulum (ER)during mitosis (Lippincott-Schwartz and Zaal 2000; Presicoet al. 2009). In contrast to mammalian cells, the Golgi bodyin yeast and plant cells does not undergo apparent morpho-logical changes (Nebenführ et al. 2000; Seguí-Simarro andStaehelin 2006; Lowe and Barr 2007). The Golgi cisternaepresent as isolated cisternae in Saccharomyces cerevisiae.The inheritance of late Golgi cisternae depends on actin andtype V myosin Myo2 (Rossanese et al. 2001; Arai et al.2008), and early Golgi membrane inheritance is linked toER inheritance (Reinke et al. 2004). Inheritance of the Golgibody has been also studied in simpler model organisms suchas Toxoplasma gondii and Trypanosoma brucei (He et al.

Handling Editor: Aki Nakano

F. Yagisawa (*) : T. Fujiwara :M. Ohnuma :H. Kuroiwa :K. Nishida :Y. Imoto :Y. Yoshida : T. KuroiwaResearch Information Center for Extremophiles,Rikkyo (St. Paul’s) University,3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japane-mail: fyagisawa@ucsd.edu

M. Ohnuma :H. Kuroiwa : T. KuroiwaInitiative Research Unit, College of Science, Rikkyo University,Toshima-ku, Tokyo 171-8501, Japan

M. Ohnuma :H. Kuroiwa : T. KuroiwaCore Research for Evolutional Science and Technology (CREST),Japan Science and Technology Agency,Gobancho, Chiyoda-ku Tokyo 102-0076, Japan

ProtoplasmaDOI 10.1007/s00709-012-0467-6

F. Yagisawa et al.

2004; He 2007). Both protests contain only a single Golgiper cell, which allowed the behavior of the Golgi body to befollowed throughout cell cycle by live imaging. The Golgibody duplicates prior to mitosis and maintains its integrityduring mitosis. Involvement of centrioles (T. gondii) orbasal bodies (T. bruceii) in Golgi inheritance has beensuggested (He 2007).

To better understand Golgi inheritance in eukaryotes, wedecided to use the unicellular red alga, Cyanidioschyzonmerolae. This alga is advantageous for studying organelleinheritance. Its cell structure is extremely simple, containingseveral vacuoles, simple ER, and only a single nucleus,mitochondrion, microbody, and chloroplast (Kuroiwa et al.1994; Miyagishima et al. 1999; Yagisawa et al. 2007;Yagisawa et al. 2012). These organelles proliferate duringcell division, a process that can be highly synchronized(~80 %) by light and dark cycles (Suzuki et al. 1994). Thegenome for C. merolae has also been completely sequencedand is one of the smallest (16.5 Mb) and simplest amongeukaryotes (Matsuzaki et al. 2004; Nozaki et al. 2007).These features allow us to study organelle dynamics invarious ways. For example, proteins involved in chloroplastor mitochondrial division have been identified by proteo-mics of organelles isolated from synchronized cell culture(Yoshida et al. 2009, 2010). In addition, a protein requiredfor vacuolar inheritance has been found by using the

expression profiles of C. merolae cell cycle (Fujiwara etal. 2009, 2010).

Several lines of evidence indicate that C. merolaelacks the conventional acto-myosin system. While thegenome encodes one actin protein, no genes encodingmyosin motor domains have been found (Matsuzaki etal. 2004). In addition, actin monomers or filaments werenot detected by immunoblot analysis or by staining withrhodamine-conjugated phalloidin or anti-actin antibodies(Suzuki et al. 1995). Furthermore, actin-depolymerizingdrugs such as cytochalasin and latrunculin had no ap-parent impact on C. merolae (Suzuki et al. 1995; ourunpublished observations). Because acto-myosin systemis often involved in organelle inheritance (Weisman2006; Fagarasanu and Rachubinski 2007), it is of greatinterest to understand how the Golgi body is inheritedin C. merolae.

While electron microscopy (EM) images have shownthat C. merolae cells have only one to two Golgi bodieswith two cisternae (Okuwaki et al. 1996), further studieshave been precluded by the technical difficulties inpreserving the Golgi structure for EM studies. To dem-onstrate Golgi inheritance, we used Golgi protein Sed5and Got1 as markers to detect the Golgi body byimmunofluorescence microscopy. Our study suggestedthe involvement of microtubules in Golgi inheritancein this alga.

Materials and methods

Cell culture and drug treatment

C. merolae strain 10D-14 (Toda et al. 1998) was maintainedin 2× Allen’s media (Allen 1959) at pH 2.5 and 42 °C undercontinuous light. For synchronization, cells were diluted toan OD444 of 1.0 and cultured under 12-h light/dark cycleswith vigorous aeration (Suzuki et al. 1994). Synchronizedcell division peaked at 2 h during the second dark period.For oryzalin treatment, 100 mM stock solution of oryzalin(Wako Pure Chemical Industries) in dimethyl sulfoxide(DMSO) (Wako Pure Chemical Industries) was added to afinal concentration of 40 μg/ml at 8 h during the secondlight period.

Transient expression of HA-tagged proteins

To generate vectors for hemagglutinin (HA)-tagged CMI302C(Got1), DNA fragments were PCR-amplified with the primers5′-tcactagttcagagatgggttcgataacac-3′ and 5′-tcattaattaataccgggagtttcggttcga-3′ using genomic DNA as a template.Amplified fragments were digested with Spe1 (Takara Bio)and Pac1 (New England Biolabs) and cloned into the

�Fig. 1 Golgi inheritance in C. merolae. a, b Co-localization of Sed5with Got1. Cells were transformed with HA-tagged Got1, fixed, andthen stained with anti-HA (HA) and anti-Sed5 antibodies. DNA wasstained with DAPI. Signals in the chloroplast regions were due tochlorophyll autofluorescence. Interphase (a) and M phase (b) cells.Sed5 labeled granular structures intensely, which colocalized with HAsignals (arrowheads) and faint punctate structures (arrows). c–h Inher-itance of the Golgi body during the cell cycle. Immunofluorescencemicroscopy was used to visualize cells stained with anti-α-tubulin andanti-Sed5 antibodies. DNA was stained with DAPI. Images of cells inG1 (c), S (d), G2 (e), and M (f–h) phases. i EM image of the Golgibody in the M phase cell. The Golgi body, nuclear membrane, andtubulin were marked by color (right panel). The Golgi-like structurewas localized close to the spindle pole, which were recognized bygathered microtubules. Schematic representations derived from thesepanels are shown on the right. j–m Spatial relationships betweenmicrotubules, mitochondria, and the Golgi body. Immunofluorescencemicroscopy was used to visualize cells stained with anti-α-tubulin,anti-porin, and anti-Sed5 antibodies. DNA was stained with DAPI.The images show G2 (j) and M (k–m) phases. Alpha-tubulin wasdetected with a secondary antibody conjugated to Alexa350 (bluefluorescence). Red fluorescence in the panels for tubulin/CP is auto-fluorescence from the chloroplast. Schematic representations derivedfrom these panels are shown on the rightmost panel. n–p Effects oforyzalin on the inheritance of the Golgi body. Cells were treated withDMSO (control, n) or oryzalin (o, p) and immunostained for α-tubulinand Sed5. Sed5 did not show bipolar localization in oryzalin-treatedcells. nuDNA nuclear DNA, mtDNA mitochondrial DNA, cpDNAchloroplast DNA, PC phase contrast, GB Golgi body, NU cell nucleus,NM nuclear membrane, MT mitochondrion, CP chloroplast, SP spindlepole, AF autofluorescence. Bars 2 μm (a, b, h, m, p) and 250 nm (i)

Inheritance of Golgi in the primitive red alga

pBSHAb-T3′ vector (Ohnuma et al. 2008). Transformation ofC. merolae was performed as described previously (Ohnumaet al. 2008; Yagisawa et al. 2009), and cells were further fixedfor immunofluorescence microscopy.

Fluorescence microscopy

Immunofluorescence microscopy was performed as describedpreviously (Nishida et al. 2004). Primary antibodies were usedat dilutions of 1:30 for rat anti-Sed5 antibodies to detect Golgibodies (Yagisawa et al. 2009), 1:100 for rabbit anti-α-tubulinantibodies (Fujiwara et al. 2009), and 1:100 for guinea piganti-porin antibodies to detect mitochondria (Fujiwara et al.2009). Alexa 555-conjugated goat anti-rat IgG (Invitrogen)was used for anti-Sed5 antibodies at a dilution of 1:100. Alexa488-conjugated goat anti-rabbit or guinea pig IgG (Invitrogen)was used for detecting anti-tubulin or anti-porin antibodies at adilution of 1:200. Goat anti-rabbit Alexa 350-conjugated IgG(Invitrogen) was used for anti-α-tubulin antibodies at a dilu-tion of 1:100. For detecting HA tag, Alexa 488-conjugatedmouse anti-HA antibodies (Invitrogen) were used at a dilutionof 1:500. For straining DNA, 1 μg/ml of 4′,6′-diamidino-2-phenylindle (DAPI) was used. Images were observed with a×100 objective and collected with a 3CCD digital camera(C7780; Hamamatsu Photonics). Alexa 555 signals were con-verted to magenta from red using Photoshop software (AdobeSystems).

Electron microscopy

Electron microscopy was performed as described by Okuwakiet al. (1996). Briefly, cells were sandwiched between copperplates, rapidly frozen in liquid propane chilled in liquid nitro-gen, and then transferred to dried acetone containing 1% (w/v)osmium at −85 °C. The samples were embedded in Spurr resin(Polyscience Inc), cut into 90-nm-thin sections, and stainedwith 3 % (w/v) uranyl acetate and 1 % (w/v) lead citrate. Allsamples were examined with a JEM-1200CX electron micro-scope (JEOL).

Results

To detect the Golgi body by fluorescence microscopy, westained the cells with antibodies previously raised againstCMN143C, a homologue of Sed5p (Supressor of Erd2Deletion 5) in budding yeast (Sed5; Yagisawa et al. 2009).Yeast Sed5p is a syntaxin-related protein involved in vesic-ular transport from the endoplasmic reticulum to the cis-Golgi. It localizes on early Golgi membrane and cytoplas-mic punctate structures (Hardwick and Pelham 1992). Bothyeast Sed5p and C. merolae Sed5 have a t-SNARE domainand a C-terminal transmembrane domain. The antibody

against C. merolae Sed5 was raised against the part exclud-ing the transmembrane domain. Cells stained with antibod-ies to C. merolae Sed5 exhibited mostly one to two intenselylabeled granular structures per cell (Fig. 1a, b—arrow-heads). Punctate signals with lower fluorescence intensitieswere also observed (Fig. 1a, b—arrows), while some cellshad more than three intensely labeled structures. We furtherexamined Sed5 signals by co-labeling other Golgi proteins.In budding yeasts, Got1p (GOlgi Transport 1) is involved invesicular transport and mainly functions at the early Golgimembrane (Wooding and Pelham, 1998; Conchon et al.1999). C. merolae CMI302C was found to be homologousto Got1p (identity, 31 %) by a BLAST search. We transient-ly expressed HA-tagged CMI302C (Got1) and then usedimmunofluorescence microscopy to visualize cells labeledwith anti-HA and anti-Sed5 antibodies. Intense signals forSed5 were closely colocalized with signals for HA-taggedproteins in both non-dividing and dividing cells (Fig. 1a, b),suggesting that these signals represented the Golgi bodyindependently of the stage of the cell cycle. To furtherexamine the Sed5 signals, we performed immunoelectronmicroscopy; however, we were unable to obtain images inwhich the structure of the Golgi body was visible, presum-ably because of the fragility and low abundance of the Golgibody in C. merolae. Using anti-Sed5 antibodies, we studiedthe localization of the Golgi body throughout the cell cycle(Fig. 1c–h). In C. merolae, cell cycle progression can bedetermined by the stage of organelle division (chloroplast,mitochondrion, nucleus) and the development of microtu-bule structures (Suzuki et al. 1994; Nishida et al. 2005;Imoto et al. 2010). DNAwas stained with DAPI and micro-tubules were stained with anti-α-tubulin antibodies. DuringG1 (Fig. 1c) and S phases (Fig. 1d), the Golgi body waslocated around the cell nucleus. In most G2 (Fig. 1e) and Mphase cells (Fig. 1f–h), the Golgi body was found to beadjacent to each spindle pole. The Golgi body was inheritedinto each daughter cell upon cytokinesis (Fig. 1h). No cellswith less than two Golgi bodies were observed in M phase.The number of the Golgi body per cell was, on average, 1.5±0.67, 2.05±0.67, 2.3±0.58, 2.27±0.61, and 1.41±0.65 inG1 before synchronized cell division, S, G2, M, and G1after synchronized division, respectively (n>30). By trans-mission electron microscopy, Golgi-like structures adjacentto spindle poles were observed in M phase cells (Fig. 1i).

Because spindle poles are associated with the mito-chondrial edge (Nishida et al. 2005), the detection of α-tubulin, the mitochondrial outer-membrane protein porin,and Sed5 revealed the spatial relationship between Golgibodies, mitochondria, and spindle poles (Fig. 1j–m). Nooverlap was observed; however, each organelle wasadjacent to one another in M phase, suggesting a closeinteraction between them (Fig. 1j–m). To assess whethermicrotubules are responsible for such Golgi localization,

F. Yagisawa et al.

treatment with oryzalin, an inhibitor of tubulin polymeriza-tion, was performed (Fig. 1n–p). Oryzalin treatment does notprevent chloroplast or mitochondrial division, while it inhibitsnuclear division and segregation of divided mitochondria(Nishida et al. 2005). In the presence of the drug, 87 % of Mphase cells (n0100) showed aberrant Golgi localization.Elongated Sed5 signals or two Sed5 signals localized in closeproximity to each other were observed on the nuclear surface(Fig. 1o, p). This localization was not bipolar, suggesting theinvolvement of microtubules in Golgi inheritance.

Discussion

In this study, we analyzed Golgi markers by immunofluo-rescence microscopy and demonstrated how they wereinherited. While anti-Sed5 antibody clearly labeled C. mer-olae Golgi body, faint signals were on the Got1-negativestructure. These structures could be vesicles cycling be-tween the ER and the Golgi body as is the case for yeastSed5p. Since HA-labeled Got1 was less localized to cyto-plasmic punctate structures, it may work as a better markeronce transformation efficiency is improved in C. merolae.

No M phase cells with less than two Golgi bodies werefound, indicating that Golgi proliferation occurs before mitosis,particularly when cells contain only one Golgi body. In eukar-yotes, several mechanisms of Golgi proliferation have beenproposed (Munro 2002). In mammalian cells, Golgi bodiesare disassembled and reconstructed. In yeasts and T. brucei(Glick and Nakano 2009; Bevis et al. 2002; He et al. 2005),Golgi bodies are generated de novo. De novo synthesis isfurther classified by the role of the old Golgi body in theconstruction of the new. For instance, Golgi bodies are gener-ated de novo at a distance from the old ones in the buddingyeast Pichia pastoris (Bevis et al. 2002), whereas in T. brucei,the Golgi body is generated next to the old one (He et al. 2005).In seed plants, green algae, and T. gondii, Golgi bodies undergofission (Noguchi 1978; Noguchi and Kakami 1999; Stahelinand Kang 2008; Pelletier et al. 2002). Although the mechanismof Golgi proliferation in C. merolae is not clear, given thephylogenic position of C. merolae, we predict that the C.merolae Golgi body also undergoes fission. Microtubules arelikely to play roles in fission and/or segregation of the Golgibodies in C. merolae, as shown by oryzalin treatment.

C. merolae Golgi inheritance may require spindle polesas suggested by the localization of the Golgi body and theoryzalin treatment. In C. merolae, spindle poles are involvedin mitochondrial segregation, which mediates the inheri-tance of other organelles such as vacuoles and microbodies(Miyagishima et al. 1999; Yagisawa et al. 2007). Therefore,spindle poles may work as a hub to synchronize Golgiinheritance with the inheritance of other organelles andchromosomes.

Some aspects of Golgi inheritance in C. merolae seem tobe conserved in other organisms. In T. gondii and T. brucei,Golgi bodies are found near the centrioles and basal bodies,respectively (He 2007). Involvement of these structures inGolgi inheritance as well as other organelles such as apico-plasts and mitochondria has been suggested (He 2007). Inaddition, a close association between the Golgi body and thecentrosome is found in many mammalian cell types (Riosand Bornens 2003), and a crucial role of the spindle in Golgiinheritance has been reported (Wei and Seemann 2009). Inseed plants and green algae, Golgi proliferation occurs priorto cell division (Noguchi 1978; Ueda 1997; Seguí-Simarroand Staehelin 2006), whereas the mechanism to link the cellcycle and Golgi proliferation is unknown. C. merolae pro-vides useful and unique tools to study organelle dynamics.Future studies will investigate the molecular basis for inher-itance by taking advantage of advances in the way to detectGolgi bodies in C. merolae.

Acknowledgments This work was supported by a research fellow-ship from the Japanese Society for the Promotion of Science (11044) toF.Y., grants from the Ministry of Education, Culture, Sports, Science,and Technology of Japan (22247007 and 22657061) to T.K., a grantfrom CREST to T.K. Y.Y. is supported by a Human Frontier ScienceProgram Long Term Fellowship. We have no financial relationshipwith the organizations that sponsored this research.

Conflict of interest The authors declare that they have no conflictsof interest.

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