Since the first observation of microfilaments in a freshwater alga, Nitella sp. (Nagai and Rebhun 1966), many papers have been published on the organization and function of actin filaments in interphase and other cell cycles in higher plants, fungi, green algae (Grolig 1990),red algae (Suzuki et al. 1995, Garbary and McDonald 1996a, 1996b), and brown algae (Karyophyllis et al. 2000).

Microfilaments are thought to play a key role in cytokinesis furrowing, while not being involved in nuclear division. Sampson and Pickett-Heaps (2001) observed microfilaments associated with chromosomes, often in paired spots, in Oedogonium sp., a green alga, and suggested that actin filaments may be a functional component of the spindle. Similar results have been reported regarding F-actin organization in Sphacelaria, a brown alga (Karyophyllis et al. 2000). Karyophyllis et al. (2000)observed actin filaments aligned with spindle microtubules during nuclear division and posited the existence of organized actin flilament-kinetochore bundles. They also suggested that actin filaments might be involved in chromosome movement as well as in cytokinesis furrowing.In the Zygnematales, cytokinesis starts at the center of the cell and moves toward the periphery in the phragmoplast and furrowing with a persistent spindle. The organization and function of actin filaments, and the relationship between actin filaments and microtubules during nuclear division has never been studied in this group.

In this study, we observed the cytoskeletal changes during cell division of the green alga Zygnema cruciatum (Vauch.) Agardh to elucidate the coordinate involvement of actin filaments and microtubules in nuclear division and chromosome movement.

Algal materials were collected from ponds in Kongju, Korea, from December, 2004 to January, 2005. The plants were washed three times with Bolds basal medium (Bischoff and Bold 1963) and kept in the same medium at 4°C, using a 12 : 12 hour light : dark cycle with cool-white fluorescent lighting (> 20 ㎛ol photons m^-2s^-1). Germanium dioxide was added to the medium (final concentration 1 mg L^-1) for 2 weeks to eliminate diatoms.

To visualize the actin cytoskeleton, Zygnema filaments were fixed for 30 min in 3.7% (w/v) formaldehyde diluted in microfilament stabilizing buffer (MFSB) consisting of 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% Triton X-100 diluted in MFSB. The Zygnema filaments were then washed three times with MFSB before being placed in the staining solution of Bodipy FL phallacidin (Molecular Probes, Eugene, OR, USA) (Wieland et al. 1983) for 3-6 h in the dark at 4°C. Bodipy FL phallacidin was prepared as a stock solution of 300 units mL^-1 in methanol and was stored at -20°C in the dark. The stock was diluted with MFSB to a final concentration of 1.5 units mL^-1. For double staining of F-actin and nuclear DNA, Bodipy FL phallacidin samples were placed in a solution of 2.5 μg mL-1 4’-6 diamidino-2-phenolindole (DAPI) for 10 s, washed with MFSB and mounted on slide glass with MFSB. Double-stained materials were observed with a model BX-50 fluorescence microscope (Olympus, Tokyo, Japan) under blue (excitation = 470 nm) and ultraviolet (excitation = 356 nm) filters for F-actin and nuclear DNA staining, respectively.

Zygnema filaments were fixed in 3.7% formaldehyde diluted with MFSB for 30 min and then rinsed three times in the same buffer. For microtubule staining, Zygnema filaments were cut with razor blade into small pieces (0.5-1 mm in length) and incubated overnight in the dark with monoclonal anti-α-tubulin antibody conjugated to 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). For double staining of microtubules and nuclear DNA, the sample stained with the flourescein isothiocynate (FITC)-conjugated antibody were placed in a solution of 2.5 μg mL^-1 DAPI for 10 s and washed with PBS. Double-stained materials were microscopically examined as described above.

For time-lapse video-microscopy, the filaments were placed on a glass slide and a coverslip was applied and sealed with a 1 : 1 : 1 mixture of Vaseline, lanolin and paraffin that had been prepared by melting on a hot plate at 70oC. The slide preparations were examined using an Olympus BX-51 microscope under the oil immersion ×20 objective lens, and the images were recorded using a Digital Imaging Time-Lapse Recorder (TCS Korea, Daejeon, Korea).

Cell division was observed using time-lapse video-microscopy. Cells were uninucleate and possessed two star-shaped chloroplasts (Fig. 1A). Cell division always proceeded simultaneously with nuclear division, with the cell wall first discerned at the cell periphery, with development towards the cell center (Fig. 1B). Vesicles involved in cell plate formation decreased with the development of the cell wall (Fig. 1C-G). Usually, chloroplast division followed nuclear division and cell plate formation (Fig. 1H).

Fluorescence localization of actin and DNA

Cytoskeletal changes were observed during cell division using FITC-phallacidin for F-actin staining. Microfilaments began to appear at the center of the division plate when nuclear division started (Fig. 2). These filaments were not visible in control cells pretreated with non-fluorescent phallacidin (data not shown). Therefore, filaments revealed using FITC-phallacidin presumably represented cytoskeletal microfilament bundles composed of F-actin.

In interphase cells, F-actin was randomly distributed in the cytoplasm near the cell surface. Normally, the actin filaments were not observed in the centers of the cells (Fig. 2A1). In prophase, the nuclear envelope was broken down; two typical forms of staining were observed in different locations of the cell. The first type of staining was a fibrous web around the nucleus, in which F-actin tightly surrounded the prophase nucleus forming a finely interwoven net-like cage as apparent in the surface view (Fig. 2B1). In the other type of staining, the F-actin was evident at the middle of the cells forming a fibril ring situated just beneath the plasma membrane (Fig. 2C1 & D1). A faint trace of protoplast-furrowing was visible in mid-

prophase cells. These protoplast-furrowing structures were tightly associated with the fibril ring (Fig. 2C1).

In metaphase cells, the chromosomes were aligned at the cell equator, and the net-like cage of F-actins ran nearly parallel with the individual chromosomes, but ultimately most formed a spindle located on the individual chromosomes (Fig. 2D1, 3A1 & B1). In this stage, the fibril rings were similar in appearance to those in prophase cells (Fig. 2B1 & C1). The diameter of the fibril ring

continuously decreased with the development of the furrow, until each cell divided into two daughter cells (Fig. 2D1-F1).

In anaphase cells, the duplicated chromosomes separated, and the spindle form of F-actins associated with the daughter chromosomes moved from the equator of the cell to spindle poles (Fig. 3C1 & D1). The spindle form of F-actins was similar in appearance to those in metaphase cells (Fig. 3E1). The F-actins were localized at the spindle pole side in late anaphase. Furrowing of the protoplast was most conspicuous at this stage. A fibril ring was not detected in the same area at anaphase.

In telophase cells, the daughter chromosomes reached to the mitotic poles, and actin filaments were observed on the daughter nuclei (Fig. 3E1 & E2). These F-actins tightly surrounded the telophase nucleus and continuously 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 irregularly (Fig. 3E1).

Cytoskeletal changes were observed during cell division using FITC-anti-α-tubulin to stain microtubules. In interphase cells, the microtubules were arranged beneath the protoplasm of the cells, most of them running nearly parallel to the cross-wall surrounding the nucleus (Fig. 4). In cells progressing from prophase to meta-

phase, microtubules were localized at the nuclear region at prophase (Fig. 4B1) but became an organized mitotic spindle at metaphase (Fig. 4B1). Interestingly, microtubules appeared between dividing chloroplasts (Fig. 4C1 & C3). Finally, after forming the new cell wall (Fig. 4D3), microtubules were re-arranged beneath the cell wall (Fig. 4D1).

The present study recorded the following observations. During interphase, actin filaments were located along the cell surface running parallel to the long axis of the cell. During prophase, a fibrous web of F-actin appeared around the nucleus and a fibril ring was formed at the division center just beneath the plasma membrane. The F-actins of metaphase cells co-aligned with spindle microtubules to contact individual chromosomes. The spindle form of the F-actin was associated with daughter chromosomes moving from the equator of the cell to spindle poles during anaphase. Finally, during telophase, F-actins tightly surrounded the nucleus. The amount of F-actin in the center of cell continuously decreased with the completion of cell division. These data provided the first comprehensive evidence suggesting a coordinate involvement of microfilaments and microtubules during nuclear division in Z. cruciatum.

The F-actins apparent during interphase appeared in the cytoplasm near the surface of the cell, and disappeared with the progress of cytokinesis. They might be involved in vesicle and organelle transport, either alone or with the microtubules. They could also be responsible for cytoplasmic streaming, although this function was not evident in Zygnema. The latter activity is usually generated by the sub-cortical actin filaments (Shimmen and Yokota 1994). Similar roles have been reported for F-actin systems in other green algae (Menzel and Schliwa 1986, Goto and Ueda 1988, Grolig 1990), in some red algae (Garbary and McDonald 1996a) and in some brown algae (Karyophyllis et al. 2000). Kim et al. (2005) reported phototaxis of the filamentous green algae Spirogyra spp. and suggested that coordination of actin filaments with microtubules on cell surface might be the mechanical effector of the filament movement.

Four types of microfilaments have been reported in 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 plasmalemma running parallel to the cell's long axis, located at the edges of the chloroplasts, and surrounding the nucleus. Among them, types 2 and 4 are similar to the F-actin type in Zygnema and in Oedogonium spp. (Sampson and Pickett-Heaps 2001). F-actins surrounding the nucleus were also presently associated with spindle forming microtubules in the prophase stage, which might suggest that the actin filament helps in the formation of mitotic spindles as well as the attachment of spindle fibers to the chromosomes in prophase. In many higher plant cells, as well as in some algae, the actin filaments show a particular organization during mitosis and cytokinesis, similar to that of the microtubule spindle (Panteris et al. 1992, Czaban and Forer 1994). It is important to note that actin has never been reported in the spindle of vegetative meristematic angiosperm cells (McCurdy and Gunning 1990, Liu and Palevitz 1992, Hepler et al. 1993, Cleary and Mathesius 1996). On the contrary, the actin filaments seem to participate in the spindle in the cultured callus cells of higher plants (Seagull et al. 1987, Traas et al. 1987), cells of the pteridophyte Adiantum (Panteris et al. 1992), and cells of Haemanthus endosperm (Schmit and Lambert 1987, 1990). Although similar F-actins type have been reported in brown algae Sphacelaria (Karyophyllis et al. 2000), this is the first report of F-actins in the metaphase stage of Zygnematales.

The organization of F-actin in the spindle of Zygnema cells persisted through all stages of mitosis, suggesting that F-actins may be necessary for spindle organization. The prophase actin filament cage may be involved in the maintenance of the nuclear materials after the degeneration of the nuclear envelope during the first mitotic stages. F-actins might also be involved in daughter chromosome separation, because these spindle forms of F-actin associated with daughter chromosomes move from the equator of the cell to the spindle poles during anaphase stage. Although the evidence against the involvement of actin filaments in mitosis is considerable, several studies showed the involvement of actin filaments in chromosome movement (e.g., Sampson and Pickett-Heaps 2001). Sampson et al. (1996) showed that treatment of the freshwater algae Oedogonium spp. with the actin inhibitor cytochalasin D blocks chromosomal attachment to the spindle, which prevents cells from entering anaphase. Our data also provide evidence that actin filaments might be involved in nuclear division and chromosome movement in Z. cruciatum.