The freshwater ciliated protozoan, Paramecium multimicronucleatum,usually possesses two contractile vacuole complexes (CVCs). The number of CVCs in a single cell, however, may vary from 1 to 7. We found that the number of cells that have more than two CVCs increased after the cells were exposed to a hypo-osmotic or a high Ca2+ condition. It is assumed that the biological significance of this increase in the number of CVCs is to enhance the cell's ability to eliminate excess water or Ca2+ from the cytosol. An extra CVC was either generated de novo in the posterior region of the cell or, when in the anterior region, by binary fission of the anterior CVC. Generation of these extra CVCs was not inhibited by aphidicolin,a potent inhibitor of DNA synthesis in the micronuclei of Paramecium,even though normal duplication of the CVC that accompanies normal cell division was completely inhibited by this inhibitor. These results suggest that generation of extra CVCs is controlled by a hypothetical regulatory mechanism that is activated either by a hypo-osmotic or by a Ca2+-rich condition and that differs from the regulatory mechanism that governs normal CVC duplication during cell division.

In the freshwater protozoan, Paramecium multimicronucleatum, the osmolarity of the cytosol is always higher than that of the external environment (Kitching, 1956; Stock et al., 2001). Water,therefore, constantly enters the cell through the plasma membrane by diffusion. The cell possesses osmoregulatory organelles, the contractile vacuole complexes (CVCs), which segregate excess cytosolic water and discharges it to the cell's exterior(Ishida et al., 1993; Naitoh et al., 1997; Stock et al., 2001). Segregation and discharge of the cytosolic water by the CVC temporarily ceases immediately after the cell is exposed to an iso- or hyperosmotic condition,and therefore the water influx through the plasma membrane ceases. However,the cells resume their CVC activities if kept in the iso- or hyperosmotic solution (Dunham and Kropp,1973; Ishida et al.,1996). This resumption of the CVC activity is accompanied by an increase in the osmolarity of the cytosol over that of the external solution,and therefore the external solution is no longer iso- or hyperosmotic(Stock et al., 2001). It was,therefore, reasonable to assume that the CVC may also have a role in expelling some waste substances from the cytosol along with excess water. Recent direct measurements of the ionic composition of the fluid of the in vivo CV,using ion-selective microelectrodes, have revealed that excess cytosolic Ca2+ can be expelled through the CVC along with excess cytosolic water (Stock et al., 2002a,b).

Many species of Paramecium possess two CVCs in a single cell. Individual cells in a population, however, have been reported to possess three or more CVCs, i.e. P. multimicronucleatum(Powers and Mitchell, 1910; King, 1935; Allen et al., 1990), P. caudatum (Bhatia, 1923; Wichterman, 1946), P. aurelia complex (King,1954) P. ugandae(Wichterman, 1986). What factor(s) causes generation of extra CVCs has not been determined.

In the present study, we demonstrate that in P. multimicronucleatum generation of extra CVCs is enhanced by exposing the cells to either lowered osmolarity or tincreased Ca2+concentration. Furthermore, we found that generation of extra CVCs under these conditions was controlled by a mechanism that differs from the mechanism that controls normal duplication of CVCs during cell division.

### Cells

Paramecium multimicronucleatum (syngen 2)(Allen and Fok, 1988) were cultured in an axenic medium as previously described(Fok and Allen, 1979). Osmolarity and free Ca2+ concentration of the culture medium was approximately 84 mosmol l-1(Ishida et al., 1993) and less than 0.05 mmol l-1 (M. Iwamoto, R. D. Allen and Y. Naitoh,unpublished preliminary data), respectively. Cells in the mid-logarithmic growth phase were centrifuged (at approximately 120g) for 25 s to form a loose pellet. The cells were then suspended in the experimental solution, which was changed twice to wash the cells. The cells were then incubated in the same solution for an extended adaptation period. Cell culture, adaptation and experimentation were performed at a room temperature of 25±1°C, regulated by a window-type air conditioner.

### Experimental solutions

The standard saline solution for adaptation was a mixture containing (mmol l-1 final concentration) 2.0 KCl, 0.25 CaCl2 and 1.0 Mops-KOH (pH 7.0), which had an osmolarity of 4 mosmol l-1(Naitoh et al., 1997). Osmolarities of some experimental solutions were varied by adding sorbitol to the standard saline solution. In some experiments, concentrations of KCl and/or CaCl2 were varied. The osmolarity of each solution was determined by using a freezing-point osmometer. A stock solution of aphidicolin (Sigma, St Louis, MO, USA) dissolved in dimethyl sulphoxide (DMSO)at 25 mmol l-1 was added to the cell suspension to inhibit cell cycle progression.

### Immunofluorescence microscopy of the CVC

For counting the number of CVCs in Paramecium, cells were fixed for 30 min in formaldehyde (3% in 50 mmol l-1 phosphate buffer, pH 7.4) and permeabilized for 20 min in cold (-20°C) acetone before treatment with a monoclonal antibody (mAb) raised against the smooth spongiome (SS-1 mAb, IgM) (Ishida et al.,1996). This was followed by treatment with fluorescein-isothiocyanate (FITC)-conjugated rabbit anti-mouse IgM. The cells were observed by using a Zeiss microscope equipped with epifluorescence illumination and a filter appropriate for FITC (B-2E, Nikon, Tokyo, Japan). Images of the cells were taken on Kodak Tri-X film. The number of CVCs per cell (NCVC), in more than 300 cells, was determined for each experiment.

### Frequency distribution of the number of CVCs in a single cell(NCVC) in cells growing in axenic culture medium compared to cells adapted to a 4 mosmol l-1 standard saline solution

The frequency distribution of P. multimicronucleatum cells with different NCVC growing in an axenic culture medium (84 mosmol l-1) was compared to the distribution of cells in a population of Paramecium cells adapted for 24 h to a 4 mosmol l-1 standard saline solution. As is clearly shown in Fig. 1A (black columns),approximately 79% of the cells growing in the culture medium possessed the normal number of two CVCs. Less than 10% of the cells possessed three CVCs. In contrast, the number of cells with two CVCs(Fig. 1B1) decreased to approximately 51%, while that with three CVCs(Fig. 1B2) increased to approximately 43% after the cells were adapted to the standard saline solution(Fig. 1A, white columns). Some cells in both cell populations possessed CVCs of more than three while a few cells possessed only one CVC. The maximum NCVC so far observed was seven (Fig. 1B3).

### Frequency distribution of the number of radial arms in a single CVC(NRA) in cells growing in axenic culture medium compared to cells adapted to a 4 mosmol l-1 standard saline solution

CVCs of cells adapted to a 4 mosmol l-1 standard saline solution for 24 h differed from those growing in axenic culture medium not only in NCVC but also in the number of radial arms in a single CVC(NRA). NRA was larger in cells adapted to the saline solution than those in the axenic culture medium. Fig. 2 shows the frequency distributions of NRA for the two groups of cells, growing in the culture medium (black columns) or adapted to the standard saline solution (white columns). The mean NRA for cells adapted to the standard saline solution was 11.2±2.2 (N=135) and was significantly (t-test, P<10-18) larger than that for the cells in the axenic culture medium (9.0±1.5; N=141). Moreover, NRA exhibited a wider distribution (variance s=4.8) in cells adapted to the saline solution than in cells in the axenic culture medium (s=2.3). In addition, the radial arms of the cells adapted to the standard saline solution frequently exhibited branching (Fig. 1B2; white arrowhead).

### Change in the number of cells with extra CVCs after transferring the cells into saline solutions that have different osmolarities from that of the axenic culture medium

The time course of change in the number of cells that possess three or more CVCs (the cells with extra CVCs) was determined after transferring the cells from their culture medium to one of three saline solutions with different osmolarities 4, 64 or 144 mosmol l-1, respectively. Fig. 3 shows the number of cells with extra CVCs as a function of time after the transfer. The number increased with time in all cases, even when the osmolarity was higher than the axenic culture medium (84 mosmol l-1). The increase was the highest in the solution with the lowest osmolarity, i.e. 4 mosmol l-1.

### Change in the number of cells with extra CVCs after transferring the cells into saline solutions with different K+ or Ca2+concentrations

In order to investigate external factors other than the osmotic factor that affect the extra CVC generation, the time course of change in the number of cells with extra CVCs was determined after transferring the cells from their culture medium to several different saline solutions with (1) a varied K+ concentration (0.1, 2.0 or 5.0 mmol l-1) at a constant Ca2+ concentration (0.25 mmol l-1) or (2) a varied Ca2+ concentrations (0.001, 0.1, 0.25 or 1.0 mmol l-1) at a constant K+ concentration (2.0 mmol l-1). The osmolarity of each saline solution was kept constant at 84 mosmol l-1, which equalled the osmolarity of the axenic culture medium. These cells were, therefore, not subjected to a change in the osmolarity upon their transfer into any of the saline solutions.

As is shown in Fig. 4A, the time course for the cells in each of three different solutions having different K+ concentrations was essentially identical with each other. The number increased with time from its initial value of approximately 12% to approximately 40% at 48 h.

Fig. 4B shows the time course for the cells in each of four different solutions with different Ca2+ concentrations. In each case the number increased with time and reached its plateau value at around 48 h. The plateau value was larger as the Ca2+ concentration increased.

### Change in the number of cells with extra CVCs after returning cells to axenic culture medium

In order to determine how the number of cells with extra CVCs changes after the cells are returned to the culture medium from a saline adaptation solution, a sample of cells obtained from the axenic culture was first transferred into a 4 mosmol l-1 saline solution and kept immersed in this solution for 18 h. The cells were then returned to fresh axenic culture medium and kept immersed in this solution for 60 h. Thenumber of cells that possess extra CVCs was determined for cells obtained from the sample at intervals of time during this 60 h. The cell density was also determined at the same time and presented as a percentage of the density of cells at -18 h,corresponding to the time when the cells were transferred into the 4 mosmol l-1 saline solution.

As shown in Fig. 5, the number of cells with extra CVCs (solid circles) that had increased to approximately 45% during the adaptation of the cells to the saline solution(from -18 to 0 h) did not change during the first 12 h after returning the cells to the axenic culture medium. The cell density(Fig. 5, open circles) was also unchanged during this same period. The number began to decrease between 12 and 24 h and continued to decrease. By 60 h the number had returned to its initial value of approximately 12%. The cell density had begun to increase by 24 h and continued to increase to the end of the 60 hexperiment.

### Locations in cells where extra CVCs are generated

In a normal cell division cycle, each daughter' CVC will be formed anterior to a pre-existing mother' CVC immediately before the start of cell division (King, 1935; Allen et al., 1990). Paramecium cells, therefore, typically have four CVCs just before cytokinesis occurs (Fig. 6A).

On the other hand, as described above, under a condition of either lowered osmolarity or higher Ca2+ concentration or both, some non-dividing cells formed one extra CVC at either one of two different locations. Firstly,a new CVC could be generated in the posterior region of the cell posterior to the posterior CVC. This newly generated CVC frequently seemed to be connected to a long radial arm of the mature posterior CVC(Fig. 6B). Secondly, a new CVC could also be generated by the apparent binary division of the anterior CVC(Fig. 6C). The extra CVC formed by binary division was similar to its mother CVC in both size and shape.

### Effects of aphidicolin on the extra CVC generation

To determine whether extra CVC generation is related to germinal DNA synthesis in the micronuclei, the effect of aphidicolin, a potent inhibitor of DNA synthesis in the micronuclei of Paramecium(Sabaneyeva et al., 1999), as well as in the nuclei of other eukaryotic cells, on extra CVC generation was examined. Two groups of cells obtained from the axenic culture were transferred, one into a 4 mosmol l-1 standard saline solution and the other into the same saline solution containing 25 μmol l-1aphidicolin. The numbers of cells that had 1, 2, 3, 4 or 5 or more CVCs,respectively, were determined for these two cell groups after they had been adapted to the respective saline solutions for 18 h. As is clearly shown in Fig. 7, the frequency distribution of cells with different NCVC was identical between these two cell groups.

### Factors that cause an increase in NCVC and their physiological significances

Most P. multimicronucleatum cells in an axenic culture medium have two CVCs, although some do have more than two CVCs (extra CVCs)(Fig. 1A, black columns). We found that the number of cells with extra CVCs increased while that of the cells with two CVCs decreased, after the cells were transferred into a 4 mosmol l-1 standard saline solution(Fig. 1A, white columns). Transferred cells encounter both (1) a large decrease in the external osmolarity and (2) changes in the external chemical factors, since the osmolarity of the saline solution (4 mosmol l-1) is far lower than that of the axenic culture medium (84 mosmol l-1; Ishida et al., 1993) and the chemical composition in the saline solution is much simpler than that in the axenic culture medium. The major cation species in the saline solution are K+ (2.0 mmol l-1) and Ca2+ (0.25 mmol l-1).

The number of cells with extra CVCs increased when the cells were transferred into a series of saline solutions with decreasing osmolarities but in which the ionic compositions remained the same(Fig. 3). This implies that lowering the external osmolarity is one factor that influences the extra CVC generation.

However, it was also observed that the number of cells with extra CVCs also increased slightly in a 144 mosmol l-1 saline solution(Fig. 3). This osmolarity is higher than the osmolarity of the culture medium (84 mosmol l-1),and the result implies that external factors other than lowering the osmolarity can also influence the mechanism for generation of extra CVCs.

The time course required for a population of cells to acquire extra CVCs after transfer from an 84 mosmol l-1 axenic culture medium into saline solutions also at 84 mosmol l-1, but with different concentrations of K+ (0.1, 2.0 or 5.0 mmol l-1) or Ca2+ (0.001, 0.1, 0.25 or 1.0 mmol l-1), clearly demonstrated that generation of extra CVCs was essentially unaffected by varying the external K+ concentration(Fig. 4A), while it was enhanced by increasing the external Ca2+ concentration(Fig. 4B). This implies that an increase in the external Ca2+ concentration is a second factor that promotes the extra CVC generation. This hypothesis may account for the increase in the number of cells with extra CVCs found in 144 mosmol l-1 saline solutions (Fig. 3; filled circles) which, in fact, did have higher Ca2+concentrations than the axenic culture medium, since the free Ca2+concentration in the axenic culture medium we used was less than 0.05 mmol l-1 (M. Iwamoto, R. D. Allen and Y. Naitoh, unpublished preliminary data) while that in the standard saline solution was 0.25 mmol l-1.

It was also expected that more Ca2+ ions would enter the cell through Ca2+ channels in the cell surface membrane when the cells were in the standard saline solution than in the axenic culture medium. Voltage-dependent Ca2+ channels(Naitoh and Eckert, 1968; Naitoh, 1979; Ehrlich et al., 1984; Machemer, 1988) have been reported to be present in the ciliary membrane of Paramecium, while mechano-sensitive Ca2+ channels(Naitoh and Eckert, 1969) are found in the somatic membrane (Dunlap,1977; Ogura and Takahashi,1976; Ogura and Machemer,1980). Elimination of excess Ca2+ from the cytosol is extremely important for the cell to be able to maintain its Ca2+-mediated intracellular signaling systems. The cytosolic free Ca2+ concentration in the Paramecium cell was assumed to be around 10-8 mol l-1 by Naitoh and Kaneko(1972). More recently Plattner's group determined the cytosolic Ca2+ concentration of Paramecium to be 5-8×10-8 mol l-1, using a fluorochrome analysis method (Klauke and Plattner, 1998; Plattner and Klauke, 2001). Many cells have Ca2+-pumps(Ca2+-ATPases) in their plasma membranes to eliminate excess cytosolic free Ca2+ ions (Edes and Kranias, 2001). In P. multimicronucleatum a Ca2+-pump has not yet been identified in the plasma membrane;however, we recently found that the CVC of P. multimicronucleatum can accumulate as much as 20 mmol l-1 Ca2+(Stock et al., 2002b). Such a high concentration of Ca2+ in the CVC suggests that the CVC plays an important functional role in segregating excess cytosolic Ca2+and subsequently eliminating it from the cell. To achieve Ca2+regulation we now find that the Paramecium cell can also generate extra CVCs under conditions of high external Ca2+concentration.

Ca2+-pumps have been reported in the CV membrane of Dictyostelium, and have a high homology to other P-type pumps(Moniakis et al., 1995), so that Ca2+ transport into the CV by this pump may require a proton gradient between the cytosol and the inside of the CV(Moniakis et al., 1999). We have demonstrated that numerous proton pumps are indeed present in CV membranes of Paramecium (Fok et al., 1995). Heuser et al.(1993) demonstrated V-ATPases are also found in the CV membrane of Dictyostelium. Thus CVCs in Dictyostelium may also play a role not only in osmoregulation but also in Ca2+ discharge from the cytoplasm. In fact V-ATPases seem to be a constant component of contractile vacuole systems as they have also been shown in the tiny CVs of Phytophthora zoospores(Mitchell and Hardham, 1999). We now need to see if there is a functional link between proton pumps and Ca2+ uptake in the CVCs of Paramecium.

Another finding is that the number of radial arms in a single CVC(NRA) is larger in cells adapted to a 4 mosmol l-1 standard saline solution than those in cells in the axenic culture medium (Fig. 2). It can be assumed that an increase in NRA will enhance the rates of segregation of both excess cytosolic water and Ca2+ in a cell in addition to an increased NCVC.

### Cell division normalizes NCVC

As is clearly shown in Fig. 5, the number of cells with extra CVCs that increased during adaptation of the cells to a 4 mosmol l-1 saline solution started to decrease to its normal value (approximately 12%) as the cells began to multiply, approximately 24 h after the cells were returned to a normal axenic culture medium. This implicates cell division in the reduction of NCVC. It can, therefore, be expected that NCVC might increase, if cell division is inhibited. In fact, a slight increase in the number of cells with extra CVCs was observed in an 84 mosmol l-1 saline solution containing a very low Ca2+ concentration (0.001 mmol l-1; Fig. 4B, filled triangles). Upon transferring into this solution, the cells encountered neither a lowering of the external osmolarity nor an increase in the external Ca2+concentration, but they did encounter a deletion of the external nutrients that are necessary for cell division (Fig. 5; open circles).

### Extra CVC generation is governed by a mechanism(s) that differs from duplication of CVCs during normal cell division

We clearly demonstrated that extra CVC generation was enhanced by transferring the cells into hypo-osmotic or Ca2+-rich saline solution (Figs 1, 3, 4) but in which normal cell division stopped (Fig. 5). It is known that during normal cell division, the appearance of new daughter CVCs is followed immediately by the first signs of the formation of a division zone across the middle of the cell (Fig. 6A) (Allen et al.,1990; Fok et al.,2002). It is therefore suggested that extra CVC generation is independent of cell cycle progression. In fact, addition of aphidicolin, which inhibits micronuclear DNA synthesis in Paramecium(Sabaneyeva et al., 1999), had no effect on the generation of extra CVCs(Fig. 7). This supports the hypothesis that extra CVC generation is independent of micronuclear DNA synthesis on which normal CVC duplication cycles depend.

In addition, the manner in which extra CVC generation occurs differs morphologically from that of daughter CVC generation during normal cell division. The two daughter CVCs always arise anterior to their mother CVCs(King, 1935; Kaneda and Hanson, 1974; Allen et al., 1990) at nearly the same time in the cell cycle, so that cells showing signs of a division furrow almost always have four CVCs (Fig. 6A). On the other hand, in many cells one extra CVC is generated posterior to the posterior CVC (Fig. 6B) or an extra CVC can be generated by the division of the anterior CVC (Fig. 6C). These morphological differences also strongly support the idea that the mechanism(s)that governs the generation of the extra CVCs differs from that which governs generation of new CVCs during cell division.

We thank Marilynn S. Aihara for providing expert technical assistance. This work was supported by NSF grants MCB 98 09929 and MCB 01 36362.

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