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kfv. Space Res. Vol. 28,No. 4,pp. 549-553.2001 C 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved

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DIFFERENTIATION OF DICTYOSTELIUM DISCOIDEUM VEGETATIVE CELLS INTO SPORES

DURING EARTH ORBIT IN SPACE

A. Takahashi’, K. Ohnishi’, S. Takahashi’, M. Masukawa’, K. Sekikawa2, T. Amano2, T. Nakano’, S. Nagaokaj, and T. Ohnishi’#

‘Y

‘Department of Biology, Nara Medical University, Kashihara, Nara 634-8521, ace Experiment Department, National Space Development Agency of Japan, Tsukuba, Ibaraki 305-0047,

Department of Gravitational Physiology, Fujita Health University School of Health Sciences, Toyoake, Aichi, 470-l 192, Japan

# tohnishi@naramed-u.ac.jp / Fax:+81-744-25-3345

ABSTRACT

We reported previously that emerged amoebae of Dictyosterium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, ys 13, and the parental strain, NC4. In ~~13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

INTRODUCTION 0 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

The most important features of the space environment with respect to biology are microgravity and space radiation. It is important to understand the biological effects of space radiation on organisms in order to protect the health of crews when they must stay in space for a long time, because space radiation contains more ionizing radiation and high LET particles than the radiation at the surface of the earth. The National Space Development Agency of Japan (NASDA) performed the Space Radiation Environment Measurement Experiment in May 1997. The Real-time Radiation Monitoring Device (RRMD) on-board the space shuttle Atlantis (STS-84), which was launched on May 15, was used for the experiment performed on 24 May 1997. These experiments were planned for the purpose of applying its results to aim to protection of space radiation in the International Space Station (ISS), and the experimental design involved monitoring the space radiation in an orbital environment similar to that of the ISS. In addition, microgravity I ray influence the movement, spread of chemicals and chemical reaction. To test these effects, many kinds of biological experiments concerning cell differentiation, cell development from eggs (Heinrich et al., 1989; Ijiri, 1995; Snetkova et al., 1995; Souza and Black, 1985) or seeds (Kordyum, 1997; Parfenov, 1988; Tripathy et al., 1996) to adults, have been done in space. There is one very interesting report that microgravity enhanced the effects of radiation on differentiation in Carausius (C. ) morosus (Bucker et al., 1986). Here, we have aimed to determine whether the amoebae can emerge from the spores of D. discoideum and then grow, aggregate and differentiate to fruiting bodies in space by using two strains of D. discoideum. A radiation-sensitive mutant, ys 13, is very sensitive to UV, ionizing radiation and DNA damaging agents, as compared with the parental strain, NC4 (Ohnishi and Nozu, 1979; Deering et al., 1972). Moreover, mutations are also easily induced in the ~~13 cells (Podgorski and Deering, 1980; Welker and Deering, 1978). This organism has been used as a model system for the study of fundamental processes in cellular differentiation. Therefore, if we find

549

550 A. Takahashi et al.

abnormal phenomena in ysl3 but not in NC4, we can assume that space radiation may induce some kinds of DNA damage in these cells. In contrast, when a particular normal phenomenon is absent in both strains, we may consider that microgravity might interfere with normal cell differentiation and alter the mutation repair mechanism. We have already reported that the space environment had little effect on their morphogenesis through out the whole life cycle from spores to fruiting bodies, or on viability or mutation frequency at the spore stage in space (Ohnishi, et al., 1997; Takahashi et al., 1997). On the earth, it has been established that treatment with X-rays, UV irradiation or DNA-damaging agents induces the inhibition of cell growth and abnormal morphogenesis (Ohnishi and Nozu, 1979; Ohnishi, 1988). In this report, we describe the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in ysl3 and NC4 strains.

MATERIALS AND METHODS

Organisms. A strain of Escherichia coli Bs-~ was used as food to culture slime mold amoebae. The radiation-sensitive mutant of D. discoideum, 3,s13, was isolated from the parental wild-type strain by Dr. R. A. Deering (Pennsylvania State University, USA). Preparation of Bacterial Cells. After the bacterial cells were cultured overnight in bouillon broth, the cells were washed twice with phosphate (Pi)-buffer (2.8 mM Na2HPO4, 2.2 mM KH2PO 4, pH 6.4). The bacterial cells were used within 1 month as food for amoeboid D. discoideum. For the amoebae culture on membranes, 5 x 10 ~ 0 bacterial cells were filtered in the range of 60 mm in diameter on a membrane (70 mm in diameter, cellulose nitrate membrane filter, 0.454tm pore size, black, ADVANTEC, Toyo Roshi Kaisha, Ltd, Tokyo, Japan) and dried in air for about 30 rain at room temperature, and then kept in the cold at about 5°C. Culture in Space. The cell culture kit was a set of experimental devices for cultivation of both strains in microgravity environments. One cell culture kit was taken into space on a space shuttle (STS-84) and three cell culture kits were left on earth as controls. After activation by injecting Pi-buffer into the cell culture kit, we continued the incubation for a total of 7 days at 22-23°C. For a permanent record of the experimental results, we used an 8-mm video camera (Cannon L1, Cannon, Tokyo, Japan) to record the observations 7 days after activation on board (Figure 1). Collection of Spores. After deactivation by transfer to a refrigerator for about 5 days, the cell culture kits were carried from Kennedy Space Center (Florida, USA) to Nara Medical University (Nara, Japan) at 5°C. The fruiting bodies of the two strains were removed from the cell culture kits. All cells were released from the membrane by washing with 5 ml of Pi-buffer and dispersed by agitation, and spores were manually counted in a haemocytometer chamber viewed with a phase microscope. Germination of Spores. The procedures used were described previously (Nozu et al., 1980). Spores were suspended in 0.5 ml of Pi-buffer containing 0.4% Brij 58 incubated for 5 min on ice, and then washed three times with Pi-buffer and, pelleted after each wash by centrifuging at 1000 x g for 5 rain. The suspensions of washed spores in Pi-buffer were heated at 45°C for 30 rain in a water bath, and cooled rapidly on ice for 1 minute before adjusting the temperature of the suspension to 22°C, followed by addition of 0.1 volume of Bs-~ cell suspension (2 x 10 ~° cells). The time of this addition was designated as zero time of incubation for germination. Viability was then assessed by assaying plaque formation of the spores using appropriate dilution series and plating with Bs-i cells. They were incubated at 22-23~C with vigorous shaking for the times shown in figure 3. Three different stages of cells were present, namely, spores, swollen spores and emerged amoeboid cells. These were counted with a haemocytometer under a phase contrast microscope at suitable intervals during the incubation period. Mutation Frequency. The procedures were in principle the same as those previously described (Ohnishi et al., 1982). After germination, the expression of mutation was achieved by incubation of amoeboid NC4 cells in suspension for 36 h. The methanol-resistant mutants were counted by plating with bacterial cells on nutrient agar plates (N-plates) (Bonner, 1947) containing 3% methanol. Plaques were counted daily for up to 7 days. The counts of viable cells were estimated as the number of the plaques formed by co-plating with Bsq cells on N-plates without methanol.

RE SULTS

Number and Viability of Spores. In space, each strain formed completely the normal fruiting bodies, which consists of three parts: a spore mass, a stalk and a basal disc. Any fruiting bodies of abnormality were not observed in each strain. Typical photographs of fruiting bodies are shown in Figure 1.

Differentiation of D, discoideum in Space 551

Fig. 1. Typic~d photographs of fruiting bodies. A and C, NC4; B and D, 7s13; A and B, on earth; C and D, in space. Size bar, 400 I.tm.

There were hardly any abnormal fruiting bodies in the space environment. We found almost the same dimensions, namely opposite direction to a membrane and numbers of fruiting bodies formed in space as on the ground. After collecting spores from the fruiting bodies, the number of spores per fruiting body was counted (Figure 2). In the ground control samples, NC4 and 7s13 had 1.04 x 104 +_ 0.04 x 10~and 3.78 x 103 _+ 0.76 x 103 spores per fruiting body, respectively. In NC4, the number of spores formed in space was 5 . 2 8 x 103 _+0.85x 103, about one half (p<0.01) of the number in ground controls. On theo the r hand, in ¥s13, we hardly could find any spores formed in space, though we detected spores formed on earth. The viabilities of spores formed on earth in NC4 and 7s13 were 24.2% and 20.8%, respectively, under ordinary experimental conditions (Figure 2). The viability of NC4 spores formed in space was 3.3% (Figure 2). However , we could not detect the viable ys l3 spores formed in space, because 7s13 spores were not formed in space.

A

0 ~ 1 . 5 ~ . - x ( / ) , ~

O 1.0

-Q 0.5 E " I

Z 0

t i

N.D.

.,,tX~ o e .,,tX~ o e

NC4 ys-13 Fig. 2. Number of spores formed on earth or in space per fruiting body. Closed columns, the viabile spores per fruiting body; open columns, non-viabile spores. ***, significant difference between the number of spores formed on earth and in space by the Student's t-test at p< 0.001. n=4, duplicates of the same collection of spores per group. N.D., not detected.

552 A. Takahashi et al.

Kinetics of Germination. The collected spores were treated for their germination. Appearance of amoebae from spores formed in space required to longer period about 3 times compared with the ground control in the wild type strain. In the space samples of "f s 13, amoeboid cells did not appear even two weeks after treatment for germination. Nevertheless, we detected amoebae 6 h after treatment for germination. The growth rate of NC4 amoebae derived from the spores in the space samples and the ground samples were almost the same between the space samples and the ground samples. The doubling times of NC4 and ,/s 13 amoebae on the ground were about 4 and 6 h under these conditions, respectively (data not shown). Mutation Frequency of Amoebae. The mutation frequencies of amoebae derived from spores formed in space or on earth are shown in Table 1. In NC4, there was no difference between the mutation frequency in the space-derived sample and the ground control (p>O.05). These values were within the normal range.

Table 1. Mutation frequencies of amoebae emerged from spores in NC4.

Samples

on Earth

in Space 1.02 -++ 0.09

Mutation frequency ( × 10 f')

1.03 _+_+ 0.19

Values represent means ± SD. n=5, duplicates of the same collection

of spores per group.

D I S C U S S I O N

The purpose of the present investigation was to examine the effect of the space environment, which includes space radiation and microgravity, on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space by using two strains of D. discoideum. In NC4, the number of spores formed in space was about half of the number of spores formed on the ground. The viability of spores formed in space was one-eighth to the ground control. This suggests that space environment may inhibit the viability of spores formed in space. In addition, we observed a delay in the germination of spores formed in space as compared with those formed on the ground in the case of NC4. This delay appeared to be independent of the effects of space radiation, "although the same phenomenon appeared after UV irradiation (Nozu et al., 1982; Demsar and Cotter, 1981; Hamer and Cotter, 1982) or y-ray (Deering et al., 1972). The delay (12 h) of the logarithmic growth phase appears to be caused simply by the decrease in the density of viable spores formed in space. This assumption is supported by the increase of the amoeboid cell number of the space sample after the time required for 3 cell divisions to a level equal to that of the ground control sample, assuming a doubling time of 4 h for NC4. Although it was reported that microgravity enhances the mutation frequency caused by radiation in Drosophila melanogaster (Ikenaga et al., 1997), we found no significant differences in mutation frequency between amoeboid cells of NC4 from spores formed in space and on earth. We speculate that the total amounts of space radiation may be too small to affect the mutation frequency in the slime mold. Otherwise, space radiation may induce the cell killing in the damaged cells completely, because they are high LET particles and seriously irreparable damage to DNA molecules. In "¢s13, we could not find any spores formed in space. To confirm these results, we cultured the spore preparation in the presence of bacterial cells as food for amoebae. However, in 7s13 of the space sample, amoeboid cells did not appear even two weeks later. If there were some spores (even one spore), we could have detected the amoebic cells after incubation for 1 week. Therelbre, we concluded that there were no spores in the space samples of ~,s13. In the differentiation of organisms, it has been reported that microgravity enhances the effects of radiation on abnormal development in C. morosus (Bucker et al., 1986). Moreover, on the Russian Space Station Mir, an American astronaut harvested the mature dwarf wheat grown for a few months and brought more than 300 seed heads back to the earth. However, scientists discovered that all the seed heads were empty (Sychev et al., 1998; Lucid, 1998). In the present experiments, it was shown that there was a depression of spore formation in NC4 and no spore formation in "1s13. We also found a delay of the germination of the NC4 spores formed in space compared to the ground control. On the other hand, there was no difference between the mutation frequency in the space-derived samples and the ground control in the case of NC4. Taking these results into consideration, we speculate that the space environment, combining microgravity and space radiation, may not affect cell growth and the morphology in D. discoideum, but does affect some stage(s) of development, especially cell differentiation from amoebic cells to spores.

A C K N O W L E D G M E N T S

The study was performed with the support of the joint NASA and NASDA RRMD program.

Differentiation of D. discoideum in Space 553

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