Susceptibility of Individual Cells of Saccharomyces cerevisiae to the Killer Toxin K1

Biochemical and Biophysical Research Communications 283, 526 –530 (2001) doi:10.1006/bbrc.2001.4809, available online at http://www.idealibrary.com on

Susceptibility of Individual Cells of Saccharomyces cerevisiae to the Killer Toxin K1 M. Bartunek,* ,1 O. Jelinek,* and V. Vondrejs† *Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, Prague 2, 121 16 Czech Republic; and †Department of Genetics and Microbiology, Faculty of Science, Charles University, Vinicˇna´ 5, Prague 2, 128 44 Czech Republic

Received February 28, 2001

The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions. The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin. For this purpose the rhodamine B assay in a modified form was used. In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied. Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated. Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups. The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds. The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures. © 2001 Academic Press

The killer phenomenon is quite common among yeast. Killer yeast secretes toxicant proteins, which are lethal to certain sensitive yeast strains (1). The killer strain is immune to the effects of its own toxin. Many killer systems were studied so far in various genera of yeast and yeast-like organisms: Saccharomyces, Ustilago, Kluyveromyces, Pichia, Wiliopsis, Candida, Debaryomyces, and others (2, 3). There exist many classes of killer yeast strains differing particularly in the spectrum of their activity against sensitive strains, in their cross-reactivity, genetic determination of killer toxin and killer toxin immunity, and in features and molecTo whom correspondence should be addressed. Fax: ⫹420-2-67228-220. E-mail: [email protected]. 1

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

ular mechanisms of killer toxin action. The best understood is the killer system K1 in Saccharomyces cerevisiae (2, 3). Two types of dsRNA in the cytoplasmic virus-like particles control this system. The killing mechanism of this toxin consists of two phases. The first step involves a rapid binding of heterodimeric killer protein to cell wall receptors containing 1,6-␤-Dglucan as an essential component. This step does not require energy. The second step, consisting in formation of pores in cytoplasmic membrane, on the other hand requires energy. The permeabilization of the membrane leads to a collapse of a transmembrane proton gradient, efflux of potassium ions, subsequent release of ATP and of low molecular metabolites, and finally death of the cell. The effect of the killer toxin is dependent both on its own potency and susceptibility of treated cells under selected conditions. Slowly growing, stationary or starving culture is less susceptible than the culture growing exponentially in a glucose medium (2). It appears that the effect of killer toxin on yeast membrane potential reflects physiological state dependent variation (4). The activity of killer toxin depends strongly on solution pH, presence of various ions and temperature (5). In this communication we are focused on the analysis of susceptibility to K1 toxin in individual yeast cells of sensitive strain culture using the fluorescence video microscopy as a tool. We attempt to specify which are the particular yeast cells in the population that die more preferably than others. MATERIALS AND METHODS The killer strain of Saccharomyces cerevisiae X3 was constructed by induced protoplast fusion of superkiller strain T158C and the supersensitive strain S6/1. It was used for a preparation of cell free K1 toxin solution in medium J (2). The killer toxin K1 stock solution (KTS) was prepared by the method described in (6). The modified rhodamine B assay (7) was used for both, estimating the killer toxin activity and the susceptibility of sensitive cells. The supersensitive strain was cultured under aerobic condition at 30°C to the final concentration of about 5 ⫻ 10 6 cells/ml in YEG medium (7.5

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FIG. 1. The effect of killer toxin K1 on exponentially growing culture of S. cerevisiae S6/1 in a liquid medium. (a) Dose–response curve. (b) Cell distribution in the cell groups before and after the assay. (c) Distribution of stained cells in the cell groups after the assay. Percentage of stained cells is related to the number of cells in each group. (d) Time course of the relative cell number during the assay.

g yeast extract and 10 g glucose per 1000 ml distilled water). Aliquots containing 1 ⫻ 10 7 cells were harvested by centrifugation, and suspended in 1 ml KTS properly diluted in medium J to which 0.1 ml of 0.2 M glucose was added. The samples were incubated at room temperature for 120 min, and after addition of 0.1 ml 2 ⫻ 10 ⫺3 M rhodamine B the incubation continued for further 60 min. The samples had been centrifuged and the pellets had been washed twice with distilled water before the fraction of stained cells was determined using fluorescence video microscopy. Experimental arrangements of video microscopy. To observe cells stained with rhodamine B, the excitation 546-nm and emission 600-nm transmission filters were used. Under these conditions the stained cells were brightly shining and the nonstained cells could be observed as gray ghosts. The experimental parameters (the thresholds of intensity and the background level) were fitted so that all cells could easily be classified. The software LUCIA (Laboratory Imaging, Prague) was used to control the video camera. In each measurement a sufficient number of cells (800 –1200) was photographed and counted in order to determine the distribution of cells in samples according to their size and the fractions of stained (i.e., killed) cells. The cells were divided into four groups with respect to their size: (I) Cells without bud ⫽ G1-phase cells. (II) Cells with the small buds (the diameter of the bud having less than 1/3 of parent cell diameter) ⫽ S-phase cells. (III) Cells with the medium buds (the diameter

of the bud having between 1/2 and 2/3 of the parent cell diameter) ⫽ G2/M phase-cells. (IV) Cells with the large buds (the diameter of the bud being more than 2/3 of the parent cell diameter) ⫽ M-phase cells. The fractions of stained cells in the total cell population and/or in the individual groups of cells were determined. Separation of cells by centrifugation in sorbitol density gradient. 2-ml samples of sorbitol solutions (40, 30, 20, 10, and 0% of sorbitol water solution) were gradually layered in a 20-ml centrifugation tube. A cell suspension was placed on top. After the short centrifugation the cells were distributed in the sorbitol gradient according to their size, small cells at the top and the large ones, i.e., usually those with a large bud, at the bottom. Fixing of cells in the agarose gel. A drop of a cell suspension was mixed with 1% of low melting agarose solution in distilled water at temperature about 40°C. This mixture was poured onto the tipped microscopic glass to make a thin single layer containing the yeast cells. Then photo-snaps of about 1500 cells were taken. A drop containing diluted killer toxin was added together with 2 ⫻ 10 ⫺3 M rhodamine B onto the gel. After 30 min the gel was thoroughly washed with distilled water and the photo-snaps of the same cells were taken again. Thus the fractions of stained cells in individual groups I–IV both in the beginning and end of experiment were determined.

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FIG. 2. The effect of killer toxin K1 on fractions of cell population separated by centrifugation in the sorbitol gradient. (a) The cell distribution in the cell groups before the assay. (b) Dose–response curves for a standard population and its fractions separated by centrifugation.

RESULTS AND DISCUSSION The dose–response curve shown in Fig. 1a indicates that 10% KTS kills all cells in the sample under the conditions of rhodamine B assay. With increasing concentration of KTS in the range from 0 to 10% the fraction of stained cells increased not only in the total sample (Fig. 1a), but also in each selected group of cells (Fig. 1c). Cells were grouped according to the size of their buds in the end of the assay in this case. S-phase cells appear to be the most susceptible. It should be mentioned, however, that cells from this group maybe had no bud in the beginning of the assay, when the decision for the killing occurred. In order to obtain the information about the possible transition between the groups during the assay, the cell division both in presence and in absence of killer toxin was compared (Fig. 1d). It was ascertained that cell division is partially inhibited by 2.5% KTS, but the fraction of cells that divide during the assay under this condition is still larger than the group IV in the beginning of the assay (Fig. 1b). The total fraction of cells dividing during the assay, in absence of the toxin, is almost equal to the groups III and IV. These results suggest that even in presence of the toxin at least some cells continue in their cell cycle progression. The results summarized in Fig. 1b support this conclusion and demonstrate that even after application of 10% KTS some cells have to divide, because in presence of the toxin more cells accumulate in G1 than in its absence. The accumulation in G1 increases with concentration of killer toxin, very likely because the growth of cells during the assay is inhibited in concentration dependent manner. These results show, that susceptibility of cells to the toxin

depend on their position in cell cycle. Due to the reasons discussed above, however, it is not possible to say in which phase are they most susceptible, because this arrangement of experiment cannot be exploited for obtaining the information about the susceptibility of cells in the relation to their state in the beginning of the assay. In order to clarify this point, the assay was modified. The killing effect was analyzed in fractions of the exponentially growing culture, which were separated by centrifugation in sorbitol gradient. Using this method a fraction T (top) was enriched for G1-phase cells, and fraction B (bottom) for M phase-cells (Fig. 2a). These cell-fractions were exploited for analysis of killing. Dose response curves (Fig. 2b) illustrate the fact that cells from the fraction T are more susceptible than cells from the standard exponential culture, and on the other hand, cells from B are more resistant to the killer toxin. This result supported our expectation that susceptibility of cells from different phases of cell cycle in the beginning of the assay is not the same by showing that G1-phase cells are more susceptible than M-phase cells. However, even this experiment could not be exploited for ideal evaluation of relative susceptibility of cells in individual phases of cell cycle, because only in the case of G1-phase the quality of enrichment was really perfect. Only killing the cells entrapped in the agarose gel allowed us to determine, what was the state of finally stained or unstained cells just before the killer toxin was added to the sample and in the end of the assay respectively. The results summarized in Fig. 3a clearly show, that the smaller cells in the beginning of the assay are more sensitive to the killer toxin treatment,

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FIG. 3. The effect of killer toxin K1 on cells imbedded in the agarose gel. (a) Distribution of stained cells in the cell groups before the assay. (b) Distribution of stained cells in the cell groups after the assay. (c) Distribution of cell cycle transitions during the assay. (d) Distribution of stained cells in the cell transition groups after the assay. Percentage of stained cells is related to the number of cells in each transition group.

than the largest cells, and the cells with a small bud are the most sensitive in the whole population under this condition. The maximum of distribution curve is shifted to the larger cells (Fig. 3b) when they are classified according to their size in the end of the same assay. This observation is in agreement with the ascertainment that at least some cells grow during the assay. Evolution of cell population during the assay is demonstrated in Fig. 3c. Figure 3d illustrates the susceptibility of cells in dependence on their transition between groups. In general, the fraction of stained cells, which do not change the group, is larger than the fraction of cells passing to the next group. This observation probably reflects the fact that the growth of the cells committed to death is more inhibited than the growth of the rest of the population. Lower susceptibility of larger cells in exponentially growing populations is in agreement with slower growth of cells before their division (8). The enhanced

susceptibility of S-phase cells (II) coincides with apical growth of buds, which is predominant in small buds whereas large buds tend to grow isodiametrically (9, 10). Since the porosity of the cell wall increases sharply in cultures shortly before buds became visible and is maximal during the initial stages of bud growth (11, 12), K1-toxin molecules that tend to aggregate may penetrate more easily to their membrane-targets in small-budded cells. In addition, it was ascertained that incorporation of ␤-1,6-glucan containing alkali soluble glucan increases mainly during the first half of the cell cycle in S. cerevisiae (9, 13), and binding of K1-toxin to a ␤-1,6-glucan receptor at the cell surface is a first step of the toxin attack. Preferable killing of smaller cells and particularly the cells with the small buds may be therefore caused by conjunction in accumulation of surface receptor molecules, increase in porosity of the cell wall, and enhanced growth activity at surface of cells in these phases of the cell cycle.

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ACKNOWLEDGMENTS This work was supported by grants from the Grant Agency of Czech Republic (No. 204/2000/0629), Charles University (No. 82/ 1998/B Bio), and Ministry of Education (No. J13/98:113100003).

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