Cytoskeletal Changes During Nuclear and Cell Division in the Freshwater Alga Zygnema Cruciatum (Chlorophyta, Zygnematales)

Research Article

Algae 2010, 25(4): 197-204 DOI: 10.4490/algae.2010.25.4.197 Open Access

Cytoskeletal changes during nuclear and cell division in the freshwater alga Zygnema cruciatum (Chlorophyta, Zygnematales)

Minchul Yoon1, Jong Won Han2, Mi Sook Hwang3 and Gwang Hoon Kim2,* 1Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongup 580-185, Korea 2Department of Biology, Kongju National University, Kongju 314-701, Korea 3National Fisheries Research and Development Institute (NFRDI), Mokpo 530-831, Korea

Cytoskeletal changes were observed during cell division of the green alga Zygnema cruciatum using flourescein isothiocynate (FITC)-conjugated phallacidin for F-actin staining and FITC-anti-α-tubulin for microtubule staining. Z. cruciatum was uninucleate with two star-shaped chloroplasts. Nuclear division and cell plate formation occurred prior to chloroplast division. Actin filaments appeared on the chromosome and nuclear surface during prophase, and the F-actin ring appeared as the cleavage furrow developed. FITC-phallacidin revealed that actin filaments were attached to the chromosomes during metaphase. The F-actin ring disappeared at late metaphase. At telophase, FITC-phallacidin staining of actin filaments disappeared. FITC-anti-α-tubulin staining revealed that microtubules were arranged beneath the protoplasm during interphase and then localized on the nuclear region at prophase, and that the mitotic spindle was formed during metaphase. The microtubules appeared between dividing chloroplasts. The results indicate that a coordination of actin filaments and microtubules might be necessary for nuclear division and chromosome movement in Z. cruciatum.

Key Words: anti-α-tubulin; microfilament; microtubule; nuclear division; phallacidin;Zygnema

INTRODUCTION

Since the first observation of microfilaments in a fresh- paired spots, in Oedogonium sp., a green alga, and sug- water alga, Nitella sp. (Nagai and Rebhun 1966), many gested that actin filaments may be a functional compo- papers have been published on the organization and nent of the spindle. Similar results have been reported function of actin filaments in interphase and other cell regarding F-actin organization in Sphacelaria, a brown cycles in higher plants, fungi, green algae (Grolig 1990), alga (Karyophyllis et al. 2000). Karyophyllis et al. (2000) red algae (Suzuki et al. 1995, Garbary and McDonald observed actin filaments aligned with spindle microtu- 1996a, 1996b), and brown algae (Karyophyllis et al. 2000). bules during nuclear division and posited the existence Microfilaments are thought to play a key role in cyto- of organized actin flilament-kinetochore bundles. They kinesis furrowing, while not being involved in nuclear also suggested that actin filaments might be involved division. Sampson and Pickett-Heaps (2001) observed in chromosome movement as well as in cytokinesis fur- microfilaments associated with chromosomes, often in rowing. In the Zygnematales, cytokinesis starts at the

This is an Open Access article distributed under the terms of the Received 11 October 2010, Accepted 07 November 2010 Creative Commons Attribution Non-Commercial License (http://cre- *Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] provided the original work is properly cited. Tel: +82-41-850-8504, Fax: +82-41-850-8479

center of the cell and moves toward the periphery in the staining, respectively. phragmoplast and furrowing with a persistent spindle. The organization and function of actin filaments, and the Fluorescence localization of microtubules relationship between actin filaments and microtubules during nuclear division has never been studied in this Zygnema filaments were fixed in 3.7% formaldehyde group. diluted with MFSB for 30 min and then rinsed three times In this study, we observed the cytoskeletal changes in the same buffer. For microtubule staining, Zygnema during cell division of the green alga Zygnema cruciatum filaments were cut with razor blade into small pieces (Vauch.) Agardh to elucidate the coordinate involvement (0.5-1 mm in length) and incubated overnight in the dark of actin filaments and microtubules in nuclear division with monoclonal anti-α-tubulin antibody conjugated to and chromosome movement. fluorescein isothiocyanate (Sigma-Aldrich, St. Louis, MO, USA), diluted 1 : 40 in phosphate buffered saline (PBS;

8 mM Na2HPO4, 2 mM NaH2PO4, 140 mM NaCl, pH 7.4). MATERIALS AND METHODS For double staining of microtubules and nuclear DNA, the sample stained with the flourescein isothiocynate Plant material and laboratory culture (FITC)-conjugated antibody were placed in a solution of 2.5 μg mL-1 DAPI for 10 s and washed with PBS. Double- Algal materials were collected from ponds in Kongju, stained materials were microscopically examined as de- Korea, from December, 2004 to January, 2005. The plants scribed above. were washed three times with Bolds basal medium (Bischoff and Bold 1963) and kept in the same medium at Time-lapse microscopy 4°C, using a 12 : 12 hour light : dark cycle with cool-white fluorescent lighting (> 20 µmol photons m-2 s-1). Germa- For time-lapse video-microscopy, the filaments were nium dioxide was added to the medium (final concentra- placed on a glass slide and a coverslip was applied and tion 1 mg L-1) for 2 weeks to eliminate diatoms. sealed with a 1 : 1 : 1 mixture of Vaseline, lanolin and par- affin that had been prepared by melting on a hot plate Fluorescence localization of actin and DNA at 70oC. The slide preparations were examined using an Olympus BX-51 microscope under the oil immersion ×20 To visualize the actin cytoskeleton, Zygnema filaments objective lens, and the images were recorded using a Dig- were fixed for 30 min in 3.7% (w/v) formaldehyde diluted ital Imaging Time-Lapse Recorder (TCS Korea, Daejeon, in microfilament stabilizing buffer (MFSB) consisting of Korea).

10 mM EGTA, 5 mM MgSO4, 100 mM PIPES-KOH, pH 6.9 (Traas et al. 1987). Zygnema filaments were rinsed three times with MFSB, and then placed for 30 min in 0.5% Tri- RESULTS ton X-100 diluted in MFSB. The Zygnema filaments were then washed three times with MFSB before being placed Observation of cell division using time-lapse in the staining solution of Bodipy FL phallacidin (Mo- video-microscopy lecular Probes, Eugene, OR, USA) (Wieland et al. 1983) for 3-6 h in the dark at 4°C. Bodipy FL phallacidin was Cell division was observed using time-lapse video- prepared as a stock solution of 300 units mL-1 in metha- microscopy. Cells were uninucleate and possessed two nol and was stored at -20°C in the dark. The stock was star-shaped chloroplasts (Fig. 1A). Cell division always diluted with MFSB to a final concentration of 1.5 units proceeded simultaneously with nuclear division, with mL-1. For double staining of F-actin and nuclear DNA, the cell wall first discerned at the cell periphery, with de- Bodipy FL phallacidin samples were placed in a solution velopment towards the cell center (Fig. 1B). Vesicles in- of 2.5 μg mL-1 4’-6 diamidino-2-phenolindole (DAPI) for volved in cell plate formation decreased with the devel- 10 s, washed with MFSB and mounted on slide glass with opment of the cell wall (Fig. 1C-G). Usually, chloroplast MFSB. Double-stained materials were observed with a division followed nuclear division and cell plate forma- model BX-50 fluorescence microscope (Olympus, Tokyo, tion (Fig. 1H). Japan) under blue (excitation = 470 nm) and ultraviolet (excitation = 356 nm) filters for F-actin and nuclear DNA Fluorescence localization of actin and DNA

DOI: 10.4490/algae.2010.25.4.197 198 Yoon et al. Cytoskeletal Changes in Zygnema cruciatum

A B

C D

E F

G H

Fig. 1. Time-lapse images of cell division process in Zygnema cruciatum. (A) Beginning of cell wall formation. Arrow shows the nucleus between chloroplasts. (B) Two stellate chloroplasts associated with the nucleus and the peripheral protoplasm. (C) Formation of vesicles at the center of cell. (D) New cell wall begins to develop. Arrow shows phragmoplast, the new cell wall and dividing chloroplasts. (E) Developing wall. (F) Develop- ing wall with reduced number of vesicles. (G) Number of vesicles reduced abruptly. (H) Chloroplast division starts (arrows). Scale bar represents: 10 µm.

Cytoskeletal changes were observed during cell divi- tin filaments were not observed in the centers of the cells sion using FITC-phallacidin for F-actin staining. Micro- (Fig. 2A1). In prophase, the nuclear envelope was bro- filaments began to appear at the center of the division ken down; two typical forms of staining were observed plate when nuclear division started (Fig. 2). These fila- in different locations of the cell. The first type of staining ments were not visible in control cells pretreated with was a fibrous web around the nucleus, in which F-actin non-fluorescent phallacidin (data not shown). Therefore, tightly surrounded the prophase nucleus forming a finely filaments revealed using FITC-phallacidin presumably interwoven net-like cage as apparent in the surface view represented cytoskeletal microfilament bundles com- (Fig. 2B1). In the other type of staining, the F-actin was posed of F-actin. evident at the middle of the cells forming a fibril ring situ- In interphase cells, F-actin was randomly distributed ated just beneath the plasma membrane (Fig. 2C1 & D1). in the cytoplasm near the cell surface. Normally, the ac- A faint trace of protoplast-furrowing was visible in mid-

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A1 A2 A3

B1 B2 B3

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E1 E2 E3

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Fig. 2. Phallacidin staining of Zygnema cruciatum actin during cell division. (A) Interphase. (B) Prophase. (C) Prometaphase. (D) Metaphase. (E) Anaphase. (F) Telophase. (A1-F1) Phallacidin stained actin filaments. (A2-F2) Merged pictures of Phallacidin stained actin filaments and DAPI stained nucleus. (A3-F3) DAPI stained nuclei. Scale bar represents: 10 µm.

DOI: 10.4490/algae.2010.25.4.197 200 Yoon et al. Cytoskeletal Changes in Zygnema cruciatum

prophase cells. These protoplast-furrowing structures continuously decreased with the development of the fur- were tightly associated with the fibril ring Fig( . 2C1). row, until each cell divided into two daughter cells (Fig. In metaphase cells, the chromosomes were aligned 2D1-F1). at the cell equator, and the net-like cage of F-actins ran In anaphase cells, the duplicated chromosomes sepa- nearly parallel with the individual chromosomes, but ul- rated, and the spindle form of F-actins associated with timately most formed a spindle located on the individual the daughter chromosomes moved from the equator of chromosomes (Figs 2D1, 3A1 & B1). In this stage, the the cell to spindle poles (Fig. 3C1 & D1). The spindle form fibril rings were similar in appearance to those inpro- of F-actins was similar in appearance to those in metaphase cells (Fig. 2B1 & C1). The diameter of the fibril ring phase cells (Fig. 3E1). The F-actins were localized at the

A1 A2 A3

B1 B2 B3

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Fig. 3. Phallacidin staining of Zygnema cruciatum actin during cell division. (A & B) Metaphsae. (C & D) Anaphase. The spindle form of F-actins associated with the daughter chromosomes (arrows). (E) Telophase. The F-actins tightly surrounded the telophase nucleus (arrows). (A1-E1) Phal- lacidin stained actin filaments. (A2–E2) DAPI stained nucleus. (A3-E3) Differential interference microscopic image. Scale bar represents: 10 µm.

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spindle pole side in late anaphase. Furrowing of the prophase, microtubules were localized at the nuclear region toplast was most conspicuous at this stage. A fibril ring at prophase (Fig. 4B1) but became an organized mitotic was not detected in the same area at anaphase. spindle at metaphase (Fig. 4B1). Interestingly, microtu- In telophase cells, the daughter chromosomes reached bules appeared between dividing chloroplasts (Fig. 4C1 to the mitotic poles, and actin filaments were observed & C3). Finally, after forming the new cell wall (Fig. 4D3), on the daughter nuclei (Fig. 3E1 & E2). These F-actins microtubules were re-arranged beneath the cell wall (Fig. tightly surrounded the telophase nucleus and continu- 4D1). ously decreased with the completion of cell division. Different types of F-actins were evident in the cytoplasm near the surface of the cell, most of them running irregu- DISCUSSION larly (Fig. 3E1). The present study recorded the following observa- Fluorescence localization of microtubules and tions. During interphase, actin filaments were located DNA along the cell surface running parallel to the long axis of the cell. During prophase, a fibrous web of F-actin ap- Cytoskeletal changes were observed during cell divi- peared around the nucleus and a fibril ring was formed at sion using FITC-anti-α-tubulin to stain microtubules. the division center just beneath the plasma membrane. In interphase cells, the microtubules were arranged be- The F-actins of metaphase cells co-aligned with spindle neath the protoplasm of the cells, most of them running microtubules to contact individual chromosomes. The nearly parallel to the cross-wall surrounding the nucleus spindle form of the F-actin was associated with daugh- (Fig. 4A1). In cells progressing from prophase to meta- ter chromosomes moving from the equator of the cell to

A1 A2 A3

B1 B2 B3

C1 C2 C3

D1 D2 D3

Fig. 4. Fluorescein isothiocynate (FITC)-anti-α-tubulin stained microtubulin of Zygnema cruciatum during cell division. (A) Interphase. (B) Pro- phase. (C) Metaphase. (D) Telophase. (A1-D1) FITC-anti-α-tubulin stained microtubulin. (A2-D2) DAPI stained nucleus. (A3-D3) Differential interference microscopic image. Scale bar represents: 10 µm.

DOI: 10.4490/algae.2010.25.4.197 202 Yoon et al. Cytoskeletal Changes in Zygnema cruciatum

spindle poles during anaphase. Finally, during telophase, Lambert 1987, 1990). Although similar F-actins type have F-actins tightly surrounded the nucleus. The amount of been reported in brown algae Sphacelaria (Karyophyllis F-actin in the center of cell continuously decreased with et al. 2000), this is the first report of F-actins in the meta- the completion of cell division. These data provided the phase stage of Zygnematales. first comprehensive evidence suggesting a coordinate The organization of F-actin in the spindle of Zygnema involvement of microfilaments and microtubules during cells persisted through all stages of mitosis, suggesting nuclear division in Z. cruciatum. that F-actins may be necessary for spindle organization. The F-actins apparent during interphase appeared The prophase actin filament cage may be involved in the in the cytoplasm near the surface of the cell, and disap- maintenance of the nuclear materials after the degenera- peared with the progress of cytokinesis. They might be tion of the nuclear envelope during the first mitotic stag- involved in vesicle and organelle transport, either alone es. F-actins might also be involved in daughter chromo- or with the microtubules. They could also be respon- some separation, because these spindle forms of F-actin sible for cytoplasmic streaming, although this function associated with daughter chromosomes move from the was not evident in Zygnema. The latter activity is usually equator of the cell to the spindle poles during anaphase generated by the sub-cortical actin filaments (Shimmen stage. Although the evidence against the involvement of and Yokota 1994). Similar roles have been reported for F- actin filaments in mitosis is considerable, several stud- actin systems in other green algae (Menzel and Schliwa ies showed the involvement of actin filaments in chro- 1986, Goto and Ueda 1988, Grolig 1990), in some red al- mosome movement (e.g., Sampson and Pickett-Heaps gae (Garbary and McDonald 1996a) and in some brown 2001). Sampson et al. (1996) showed that treatment of algae (Karyophyllis et al. 2000). Kim et al. (2005) reported the freshwater algae Oedogonium spp. with the actin phototaxis of the filamentous green algae Spirogyra spp. inhibitor cytochalasin D blocks chromosomal attach- and suggested that coordination of actin filaments with ment to the spindle, which prevents cells from entering microtubules on cell surface might be the mechanical ef- anaphase. Our data also provide evidence that actin fila- fector of the filament movement. ments might be involved in nuclear division and chro- Four types of microfilaments have been reported in mosome movement in Z. cruciatum. Spirogyra spp. (Goto and Ueda 1988). Each type displays a unique behavior during the cell cycle: dispersed in the cytoplasm near the cell surface, located beneath the plas- ACKNOWLEDGEMENTS malemma running parallel to the cell's long axis, located at the edges of the chloroplasts, and surrounding the nu- This work was supported by a grant (RP-2010-AQ-096) cleus. Among them, types 2 and 4 are similar to the F-ac- from National Fisheries Research and Development In- tin type in Zygnema and in Oedogonium spp. (Sampson stitute, Korea to GHK. and Pickett-Heaps 2001). F-actins surrounding the nucleus were also presently associated with spindle forming microtubules in the prophase stage, which might suggest REFERENCES that the actin filament helps in the formation of mitotic spindles as well as the attachment of spindle fibers to the Bischoff, H. W. & Bold, H. C. 1963. Phycological studies IV. chromosomes in prophase. In many higher plant cells, as Some soil algae from enchanted rock and related algal well as in some algae, the actin filaments show a particu- species. The University of Texas Publications, Austin, TX, lar organization during mitosis and cytokinesis, similar 95 pp. to that of the microtubule spindle (Panteris et al. 1992, Cleary, A. L. & Mathesius, U. 1996. Rearrangements of F- Czaban and Forer 1994). It is important to note that ac- actin during stomatogenesis visualised by confocal mi- tin has never been reported in the spindle of vegetative croscopy in fixed and permeabilised Tradescentia leaf meristematic angiosperm cells (McCurdy and Gunning epidermis. Bot. Acta 109:15-24. 1990, Liu and Palevitz 1992, Hepler et al. 1993, Cleary and Czaban, B. B. & Forer, A. 1994. Rhodamine-phalloidin and Mathesius 1996). On the contrary, the actin filaments anti-tubulin antibody staining of spindle fibres that seem to participate in the spindle in the cultured cal- were irradiated with an ultraviolet microbeam. Proto- lus cells of higher plants (Seagull et al. 1987, Traas et al. plasma 178:18-27. 1987), cells of the pteridophyte Adiantum (Panteris et al. Garbary, D. J. & McDonald, A. R. 1996a. Actin rings in cytoki- 1992), and cells of Haemanthus endosperm (Schmit and nesis of apical cells in red algae. Can. J. Bot. 74:971-974.

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Garbary, D. J. & McDonald, A. R. 1996b. Fluorescent labelling 137. of the cytoskeleton in Ceramium strictum (Rhodophy- Sampson, K. & Pickett-Heaps, J. D. 2001. Phallacidin stains ta). J. Phycol. 32:85-93. the kinetochore region in the mitotic spindle of the Goto, Y. & Ueda, K. 1988. Microfilament bundles of F-actin in green alga Oedogonium spp. Protoplasma 217:166-176. Spirogyra observed by fluorescence microscopy. Planta Sampson, K., Pickett-Heaps, J. D. & Forer, A. 1996. Cytochala- 173:442-446. sin D blocks chromosomal attachment to the spindle in Grolig, F. 1990. Actin-based organelle movements in inter- the green alga Oedogonium. Protoplasma 192:130-144. phase Spirogyra. Protoplasma 155:29-42. Schmit, A. C. & Lambert, A. M. 1987. Characterization and Hepler, P. K., Cleary, A. L., Gunning, B. E. S., Wadsworth, P., dynamics of cytoplasmic F-actin in higher plant endo- Wasteneys, G. O. & Zhang, D. H. 1993. Cytoskeletal dy- sperm cells during interphase, mitosis, and cytokinesis. namics in living plant cells. Cell Biol. Int. 17:127-142. J. Cell Biol. 105:2157-2166. Karyophyllis, D., Katsaros, C. & Galatis, B. 2000. F-actin in- Schmit, A. C. & Lambert, A. M. 1990. Microinjected fluores- volvement in apical cell morphogenesis of Sphacelaria cent phalloidin in vivo reveals the F-actin dynamics and rigidula (Phaeophyceae): mutual alignment between assembly in higher plant mitotic cells. Plant Cell 2:129- cortical actin filaments and cellulose microfibrils. Eur. 138. J. Phycol. 35:195-203. Seagull, R. W., Falconer, M. M. & Weerdenburg, C. A. 1987. Kim, G. H., Yoon, M. & Klotchkova, T. A. 2005. A moving mat: Microfilaments: dynamic arrays in higher plant cells. J. phototaxis in the filamentous green algae Spirogyra Cell Biol. 104:995-1004. (Chlorophyta, Zygnemataceae). J. Phycol. 41:232-237. Shimmen, T. & Yokota, E. 1994. Physiological and biochemi- Liu, B. & Palevitz, B. A. 1992. Organization of cortical micro- cal aspects of cytoplasmic streaming. Int. Rev. Cytol. filaments in dividing root cells. Cell Motil. Cytoskelet. 155:97-139. 23:252-264 Suzuki, H., Oiwa, K., Yamada, A., Sakakibara, H., Nakayama, McCurdy, D. W. & Gunning, B. E. S. 1990. Reorganization of H. & Mashiko, S. 1995. Linear arrangement of motor cortical actin microfilaments and microtubules at pre- protein on a mechanically deposited fluoropolymer prophase and mitosis in wheat root-tip cells: a double thin film. Jpn. J. Appl. Phys. 34:3937-3941. label immunofluorescence study. Cell Motil. Cytoskelet. Traas, J. A., Doonan, J. H., Rawlins, D. J., Shaw, P. J., Watts, 15:76-87. J. & Lloyd, C. W. 1987. An actin network is present in Menzel, D. & Schliwa, M. 1986. Motility in the siphonous the cytoplasm throughout the cell cycle of carrot cells green alga Bryopsis. II. Chloroplast movement requires and associated with the dividing nucleus. J. Cell Biol. organized arrays of both microtubules and actin fila- 105:387-395. ments. Eur. J. Cell Biol. 40:286-295. Wieland, T., Miura, T. & Seeliger, A. 1983. Analogs of phalloi- Nagai, R. & Rebhun, L. I. 1966. Cytoplasmic microfilaments din: D-Abu2-Lys7-phalloin, an F-actin binding analog, in streaming Nitella cells. J. Ultrastruct. Res. 14:571-589. its rhodamine conjugate (RLP) a novel fluorescent F-ac- Panteris, E., Apostolakos, P. & Galatis, B. 1992. The organi- tin-probe, and D-Ala2-Leu7-phalloin, an inert peptide. zation of F-actin in root tip cells of Adiantum capillus Int. J. Pept. Protein Res. 21:3-10. veneris throughout the cell cycle. Protoplasma 170:128-

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