The plasmodium of Physarum polycephalum contained 15·3 mmol Ca/kg fresh weight of sample, 11·8 mmol Mg/kg, 24·5 mmol K/kg and 1·4 mmol Na/kg. When the plasmodium was starved of food, the Ca content increased gradually up to 71·9 mmol/kg during 5 days of starvation. The concentration of other elements changed only slightly. The endoplasm contained 23·0 mmol Ca/kg, 12-6 mmol Mg/kg, 26·6 mmol K/kg and 1·7 mmol Na/kg, but these contents changed only slightly during starvation. The Ca, Mg, K and Na contents of the slime and the soluble fraction were also determined. In order to clarify where the accumulated Ca was localized, Ca in the plasmodium was precipitated with potassium pyroantimonate and examined by electron microscopy. In the starved plasmodium, the vacuoles which contained the electron-opaque precipitates and were located in the ectoplasm increased in number, compared with the unstarved plasmodium. At the same time the large electron-opaque granules in the extracellular slime increased in number. The electron-opaque precipitates were identified as Ca pyroantimonate by its susceptibility to removal by chelation with ethyleneglycol bis (β-aminoethyl ether) N, N, N′, N′-tetraacetic acid (EGTA) and X-ray microprobe analysis.
The plasmodium of the acellular slime mould Physarum polycephalum is morphologically differentiated into an ectoplasm composing an outer wall and an endoplasm filling the lumen. The outside of the plasmodium is covered with slime. Endoplasmic streaming results from the contraction of the contractile proteins residing in the ectoplasm, as reviewed by Clarke & Spudich (1977), Hitchcock (1977) and Korn (1978). Ca2+, Mg2+, and K+ have been shown in in vitro studies to be required for the poly-merization (Adelman, 1977) and the contraction (Hinssen & D’Haese, 1976) of the contractile proteins of the plasmodium. Therefore, it is interesting to know the contents of Ca, Mg and K of the plasmodium, and the localization sites of Ca in the view of possible analogies with muscle. Moreover, to study the electrophysiological properties of the plasma membrane of the plasmodium (Kuroda & Kuroda, 1980), in the hope of elucidating a mechanism such as ‘excitation and contraction coupling’ in muscle, it is necessary to know the contents of Ca, Mg, K and Na of the plasmodium. However, systematic data for these ion contents have not been available; especially, previous reports of Ca content have varied widely between 1 mmol/kg and 45·8 mmol/kg (Anderson, 1964; Hatano & Totsuka, 1972; Nagai, Ishima, Kukita & Takenaka, 1975). As for the localization sites of Ca in the plasmodium, no extensive investigation has been done, except that the existence of Ca-accumulating vacuoles has been reported (Braatz & Komnick, 1970, 1973; Ettienne, 1972).
In this paper, we report the Ca, Mg, K and Na contents of the plasmodium and the localization sites of Ca in the plasmodium. Moreover, we reveal that the Ca content changes depending upon the physiological condition of the plasmodium, and that the vacuoles work as an organelle for sequestration of excess Ca.
MATERIALS AND METHODS
Culture of plasmodium
Plasmodium of Physarum polycephalum was revived from sclerotium and cultured on sheets of wet filter paper sprinkled with pressed oatmeal in the dark at 20 °C according to the procedure of Camp (1936).
For starvation, plasmodium was transplanted onto a wide surface of non-nutrient agar (3% in distilled water). After most of the plasmodium crept out from the oatmeal, the paper was removed. This time was defined as the beginning of starvation. The plasmodium was left to spread out into a reticulum of ramifying strands which branched into very thin strands within a fanlike advancing front in the dark at 20 °C. Twice a day the plasmodium was transferred onto a new agar surface without oatmeal.
Atomic absorption spectrophotometry
Samples for analysis were taken as follows. Unfractionated plasmodium was scratched gently from the advancing front to the posterior reticulated region with an acid-cleaned cover-glass. Endoplasm was obtained by puncturing a vigorous thick strand with an acid-cleaned glass needle. Slime was scratched from the tract of the plasmodium. Plasmodium was centrifuged for 60 min at 100000 g at 4 °C immediately after sampling, and the resultant supernatant fluid was used as the soluble fraction. Organic substances were decomposed with hot nitric acid and hydrogen peroxide. The samples were finally dissolved in 0·1 N HC1 containing 0·5% lanthanum, which was added to protect Ca from interference with phosphate, and measured with a Hitachi Perkin-Elmer model 303 atomic absorption spectrophotometer.
To visualize Ca in the plasmodium, the pyroantimonate technique (Komnick, 1962; Legato & Langer, 1969; Herman, Sato & Hales, 1973) was employed. In order to get an acceptable compromise between ideal fine-structural preservation and maximal Ca retention, it was modified as follows. A tiny fragment of plasmodium was briefly prefixed with 4% glutaraldehyde in 0·1 M potassium cacodylate buffer, pH 7·4, and then fixed with 0·5% OsO4 containing 2 % K[Sb(OH)J, pH 7·4. After rapid dehydration in a graded chilled ethanol, the sample was embedded in Epon 812, and sectioned with a glass knife. Thin sections were stained with uranyl acetate and lead citrate, and examined with a JEM-7 electron microscope.
Other materials and methods
To examine the susceptibility of the electron-opaque precipitates to EGTA, a grid bearing unstained thin sections was floated upside down on the surface of 5 mM EGTA, pH 7·4 for 20 min at 50 °C, then stained, and examined with the electron microscope.
X-ray microprobe analysis of the electron-opaque precipitates was performed in a Hitachi H-500 transmission electron microscope equipped with a Kevex energy-dispersive X-ray analyzer. This analysis was kindly carried out by Dr H. Ishikawa, using the instruments of Hitachi Industry.
The contamination with Ca of reagents was from o μM for 0’5% OsO2 to maximum 50 μM for 3 % agar, as measured by atomic absorption spectrophotometry.
Effect of starvation on Ca, Mg, K and Na contents
Table 1 shows the contents of Ca, Mg, K and Na of the unfractionated plasmodium, the endoplasm, the slime and the soluble fraction of the unstarved plasmodium, and the changes of Ca, Mg, K and Na contents of all the fractions during starvation. The contents of all elements were determined by atomic absorption spectrophotometry, and showed good reproducibility. Ca and Mg in the soluble fraction were considerably less than Ca and Mg in the unfractionated plasmodium or the endoplasm, implying that most of the Ca and some of the Mg might be bound to sedimentable macromolecular substances and/or stored in some organelle. As for K and Na, such a difference was not observed, suggesting that K and Na existed as free ions.
Prolongation of starvation resulted in a gradual increase of the Ca content of the unfractionated plasmodium, but not of other fractions. The contents of other elements changed only slightly. This result indicates that the plasmodium accumulates specifically only Ca, and that the accumulated Ca must be bound to some macromolecular substance and/or stored in some organelle predominantly located in the ectoplasm. Here, it should be pointed out that the plasmodium decreased in mass gradually during starvation, reaching a quarter to one fifth of its original mass after 5 days. Accumulation of Ca during starvation could therefore be explained by the decrease of mass and the retention of Ca, with other elements being excreted in proportion to the decrease of mass of the plasmodium.
Ca in the plasmodium was visualized electron microscopically by precipitating with K[Sb(OH)6], In both starved and unstarved plasmodia, distinct and massive electron-opaque precipitates were consistently localized at the following sites: (a) within some types of vacuole, which contained a large concretion (Figs. 1, 3), a number of small particles associated with a matrix substance (Figs. 1, 2) or various sizes of granules (Fig. 4); (b) in mitochondria (Figs. 1, 5); (c) in the cytoplasm outside any organelle (hereafter, the term ‘cytoplasm’ is used with this meaning) (Figs. 1, 6, 7); (d) along the inner surface of the membrane (Fig. 6); and (e) in the extracellular slime in surface invaginations (Fig. 5) and adhering to the outside of the plasmodium (Fig. 1). Starved and unstarved plasmodia differed in that the vacuoles containing electron-opaque precipitates in the ectoplasm and large electron-opaque granules in the extracellular slime were much more abundant in the starved plasmodium (Fig. 1) than in the unstarved one (Fig. 7).
This result indicates that vacuoles increase in number noticeably in the ectoplasm during starvation, and that the excess Ca is stored mainly in these vacuoles. The vacuole seems therefore to be an organelle that sequesters excess Ca in order to control the Ca content of the cytoplasm. The presence of a number of large electron-opaque granules in the slime is contradictory to the observation in the preceding section, which suggested that the slime contained only trace amounts of Ca. This is discussed later.
Identification of electron-opaque precipitates
Pyroantimonate can bind and be precipitated with cations other than Ca (Komnick, 1962; Legato & Langer, 1969; Lane & Martin, 1969; Klein, Yen & Thureson-Klein, 1972), although at higher concentrations (Klein et al. 1972). This makes necessary an analysis of the chemical nature of the precipitates. This was carried out by the following methods. The electron-opaque precipitates were not the artefactual product of lead-staining alone, because they were not observed in plasmodia fixed without K[Sb(OH)8] (Fig. 8).
The presence of Ca in the precipitates was demonstrated by examining the effect of EGTA, a specific Ca-chelating agent (Figs. 9–11). The precipitates which had been found in the vacuoles (Figs. 9,10), in the invagination (Fig. 9), and outside the plasmodium (Fig. 9) were invariably solubilized, and replaced by empty holes in the section. The small precipitates along the inner surface of the membrane (Fig. 11) and in the cytoplasm (Fig. 11) were also solubilized. Intra-mitochondrial precipitates were reduced in amount. It seems unlikely that pyroantimonate forms a chelating complex with EGTA under the conditions employed, since mixing of K[Sb(OH)6] with EGTA brought about a negligible decrease of pH, compared with the case of Ca + EGTA. Furthermore, the electron-opaque precipitates were analysed by X-ray microprobe (Fig. 12). The result suggests the presence of Sb and probably Ca in the precipitates contained in the vacuoles, in the intra-mitochondrial precipitates, in the large granules located in the extracellular slime and in the small precipitates in the cytoplasm. Signals for Ca and Sb were not detected in cytoplasm that did not contain such precipitates.
All the above results are considered to indicate that Ca and pyroantimonate are the primary constituents of the electron-opaque precipitates.
The important finding reported here is that when the plasmodium is starved, Ca is accumulated mainly in the vacuoles. The number of Ca-containing vacuoles increases mainly in the ectoplasm during starvation. This finding suggests that the vacuoles may control the Ca concentration of the cytoplasm by sequestering excess Ca and possibly releasing it later. We have further studied this question on the analogy of muscle, and will publish the findings elsewhere. The existence of a number of the large Ca-containing granules in the extracellular slime of the starved plasmodium appears not to be consistent with the result of chemical analysis that showed that the slime contained only trace amounts of Ca even in the starved plasmodium. One possible explanation for this discrepancy is as follows. The slime for chemical analysis was collected from the tract far behind the plasmodium. If the large Ca granules were water-soluble, Ca would have diffused away into the agar before sampling.
On the basis of the obtained results, we can propose a hypothetical mechanism for the control of Ca concentration of the cytoplasm during starvation. When starved, the plasmodium gradually decreases in mass. This may bring about a gradual rise of Ca concentration in the cytoplasm. This increasing Ca is sequestered into the vacuoles, whose number increases primarily in the ectoplasm. Some portions of Ca accumulated in the vacuoles are secreted as large granules into invaginations of the plasma membrane and then liberated outside the plasmodium. Secreted Ca diffuses away ultimately into the agar. It is possible that Fig. 13 shows the moment of secretion of the large granules.
The finding that the Ca content changes depending on the physiological condition of the plasmodium could be one explanation for the wide variation in earlier data for Ca content (Anderson, 1964; Hatano & Totsuka, 1972; Nagai et al. 1975).
This work was supported by a research grant from the Ministry of Education of Japan. We thank Dr S. Hatano for providing the plasmodium of Physarum polycephalum and his helpful discussion, Dr H. Ishikawa for carrying out X-ray microprobe analysis and his helpful suggestion, and Dr C. lida for his technical guidance on atomic absorption spectrophotometry. We are grateful to Dr S. Higashi-Fujime for her technical guidance on electron microscopy and her helpful discussion, and to Dr C. W. Slayman for her critical reading of the manuscript and her kind advice.