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Electron probe analysis of refractive bodies in Amoeba proteus
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research 76 (1973) 31-40
ELECTRON
PROBE ANALYSIS IN AMOEBA
J. R. COLEMAN,
OF REFRACTIVE
BODIES
PROTEUS
JYTTE R. NILSSON, R. R. WARNER and P. BATT
Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642, USA and Biological Institute of the Carlsberg Foundation, Copenhagen, Denmark
SUMMARY Phospholipid organelles, termed refractive bodies, occur in the cytoplasm of Amoeba proteus. Electron probe analysis of individual granules in situ shows that their composition is variable, and that they are rich in calcium, magnesium, and potassium. The amount of calcium,magnesium, and potassium, both singly and in sum, is proportional to the phosphorus content. This is consistent with the view that the metals are complexed with a phospholipid component of the refractive body. These observations indicate that the refractive bodies of Amoeba proteus are homologous with the refractile granules of Tetrahymena pyriformis, and suggest that the two may play a similar role in the regulation of intracellular metal concentration.
Recently, electron probe analysis of lipid ‘refractile granules’ in the cytoplasm of Tetrahymena pyriformis indicated that they were intracellular stores of calcium, magnesium and potassium [5]. Quantitative electron probe analysis revealed that the amount of each of these elements in a refractile granule was proportional to the phosphorus content of the granule, suggesting that the metal ions were complexed with a phospholipid component [5]. It was pointed out that the equilibrium between the free and lipid bound forms of each of the ions would help determine the concentration of each ion free in the cytoplasm. Thus, the granules would serve as intracellular ‘buffers’ for these metabolically important ions. The particular advantages of such a mechanism for the intracellular regulation of calcium, an ion used to control several cell processes, were discussed with regard to the habitat and phy3-
721815
siology of T. pyriformis [5]. However, it was not known whether these organelles were restricted to just this one species or whether they could be a feature common to other protozoa. Morphologically similar organelles, termed refractive bodies, occur in the cytoplasm of Amoeba proteus [9]. Histochemical investigations showed that they are phospholipid and contain inorganic materials identified as calcium and magnesium [2, 71. This composition is superficially similar to that of the T. pyriformis granules, but it is not sufficient to establish an homology between the two organelles. Consequently, a quantitative electron probe analysis of individual refractive bodies in situ was performed to determine (a) whether the bodies of A. proteus have a composition similar to the granules of T. pyriformis, and (b) whether the amount of calcium, magnesium and potassium contained Exptl Cell Res 76 (1973)
32
J. R. Coleman et al. in the refractive bodies is proportional to the phosphorus content, as in T. pyrifoumis. The use of the electron probe eliminated the hazards of redistribution and loss associated with the isolation of organelles containing readily soluble constituents, and analysisIof individual granules permitted the detection of variations among individuals and size classes of organelles. MATERIALS
AND METHODS
Amoeba proteus, obtained from Wards Scientific, Rochester, N.Y., were cultured in the medium supplied and prepared for analysis the day after arrival. Single cells were placed on a clean, silicon disc in large drops of medium and permitted to attach to the substrate. Excess liquid was quickly removed with filter paper and cells were heat-fixed by passing the silicon disc bearing the cells through the flame of a propane torch, essentially the same procedure used previously for T. pyriformis [5]. Morphological preservation was assessedwith Leitz reflecting optics at 500x and 1 000 x magnification. Comparison of sample current and X-ray profiles was used to insure that no detectable redistribution of the elements of interest had occurred during preparation [5]. Electron probe analysis was performed on 79 granules in 5 cells from 3 different cultures according to techniques described previously [5, 61. Granules were analyzed for C, Na, K, Mg, Ca, S, Fe, Cu, Zn, Cl and P. Quantitative analysis was performed as before, using the BICEP computer program on an IBM 360/44 to correct X-ray intensities [ll]. This procedure is accurate to about il0 sb for atomic ratios and replicate measurements indicate precision is about +2 % [5].
RESULTS
Fig. 1. Sample current (SC) and X-ray images of Amoeba proteus. The sample current image (a) shows
the outline of the dried cell containing numerous bright refractive bodies mainly localized in tie cell body. The calcium Kcc X-ray image (b) shows that the largest part of the total cell calcium is contained within the refractile bodies. The grid marks cover identical areas in each image and the source of any X-ray signal can be identified in the SC image by referring to the grid. The phosphorus X-ray image (c) Expll
Cell Res 76 (1973)
The sample current (SC) image of a heat fixed Amoeba proteus appears in fig. 1. The refractive bodies appear as bright particles throughout the central main portion of the cytoplasm and also in the pseudopodia. About 800 refractile bodies are present in this cell. Other cell organelles are not distinct in the SC image because contrast is a function of local atomic number differences, and most shows that phosphorus is distributed throughout the cell, and that the refractive bodies are especially rich in phosphorus. Each grid division represents 40 ,um.
Amoeba proteus refractive bodies
33
Fig. 2. Sample current and X-ray images of refractive bodies. (a) SC image at t ,rm/screen division. Large
‘core’ containing bodies as well as smaller ones are visible. The smallest bodies are only faintly visible in the background. (b) a higher magnification, 1 pm/screen division, two large core-containing bodies are visible. A smaller adjacent body is indistinct in this SC image but appears clearly in the X-ray images; (c) is the calcium Kcc X-ray image of the bodies seen in (a); (b) showing that calcium is present in the granules at a higher concentration than in the surrounding cytoplasm; (d) phosphorus Ka X-ray image of the bodies in (b), showing a concentration of phosphorus in the bodies; (e) potassium Kcc X-ray image; (f) the magnesium Ka X-ray image of the same bodies, showing that the granules contain a greater concentration of each of these elements than the surrounding cytoplasm. Exptl Cell Res 76 (1973)
34
J. R. Coleman et al.
PHOSPHORUS
Fig. 3. Sample current image of refractive body at
0.5 /&m/screen division. The SC and X-ray profiles in fig. 4 were generated by driving the beam across this granule along a path indicated by the grid line marked by white triangles.
of the cell is composed of elements with similar atomic numbers. A major proportion of the total cell calcium is contained in the refractive bodies, as can be seen in the calcium X-ray image (fig. 1b). The phosphorus X-ray image (fig. 1 c) shows that while the bodies are rich in phosphorus, the surrounding cytoplasm also contains a substantial amount of this element. At higher magnifications, morphological characteristics of individual bodies can be resolved. In fig. 2a, the various types of granules can be distinguished: (1) large bodies about 1.44 pm or more in diameter, showing a central darker ‘core’; (2) intermediate spherical bodies about 1.6 pm in diameter, with no core and (3) small bodies about 0.8 pm in diameter, only faintly visible in the background at this magnification. These smallest bodies are not usually classified as refractive bodies. The solid and the coreExptl
Cell Res 76 (1973)
SAMPLECURRENT
Fig. 4. Sample current (botlom) profile of body seen
in SC image in fig. 3. The potassium Ka X-ray profile, calcium Kcc X-ray profile and phosphorus Ka X-ray profile are seen above and congruent with the SC profile.
Amoeba proteus refractive bodies 35 Table 1. Means of atomic percent for analysis of several elements in refractile granules of Amoeba proteus Figures within
parentheses are S.D. Large Edge (N=26)
Ca Mg K P
4.19 1.54 2.85 12.78
Intermediate (N=28)
Center (N= 21)
(1.29) (0.88) (1.01) (3.14)
3.23 0.75 1.48 8.54
4.04 1.09 2.76 11.63
(1.19) (0.40) (0.46) (2.51)
(1.06) (0.33) (0.78) (1.86)
Small (N=25) 3.80 1.86 3.58 14.72
(1.49) (0.61) (0.86) (3.38)
-
containing bodies are spherical in vivo, but it is not possible to determine whether they maintain this shape or whether they become discoid in the fixed cells. The three-dimensional shape of the bodies, however, does not influence the results of analysis when atomic ratios are employed. Fig. 2b shows the sample current of two large adjacent bodies. An intermediate sized body is alongside but indistinct in this mode of display. The calcium (2c), phosphorus (2d), potassium (2e), and magnesium (2f) X-ray images are presented in the samefigure. It can be seen in these images that the concentrations of each of these elements in the bodies is greater than in the surrounding cytoplasm, and that the localization of each element corresponds quite closely to the shapes of the bodies as seen in the SC image.
This is readily perceived in a comparison of SC and X-ray profiles. Fig. 3 shows the SC image of a single large core-containing body. Profiles were generatedby driving the electron beam at a constant rate in a straight line over the same path across the body. The resulting sample current or characteristic X-ray intensities were recorded on successivesweeps of the beam. These can be seen in fig. 4. The lowest trace in fig. 4 is the SC profile from the body in fig. 3 and corresponds to a trace of density across the horizontal grid line visible near the center of the image. The potassium X-ray profile in fig. 4 is directly above the SC profile and its shape is congruent with the SC profile. Above this is the calcium profile, also congruent with the SC profile. The topmost trace is the phosphorus X-ray profile, also congruent with the SC profile.
Table 2. Mean ratios of metals to phosphorus (atomic percent) in cytoplasmic granules in Amoeba proteus Figures within
parentheses are S.D. Large Edge (N-26)
Ca/P MU K/P Ca + Mg/P Ca +Mg+K P
0.332 0.119 0.225 0.450
Center (N=21) (0.074) (0.054) (0.058) (0.054)
0.676 (0.065)
0.375 0.096 0.177 0.471
(0.065) (0.060) (0.041) (0.051)
0.647 (0.072)
Intermediate (N = 28)
Small (N-25)
0.348 0.095 0.234 0.448
0.250 0.132 0.250 0.383
(0.013) (0.033) (0.046) (0.018)
0.682 (0.047)
(0.054) (0.050) (0.057) (9.038)
0.632 (0.041)
Exptl Cell Res 76 (1973)
36 J. R. Coleman et al.
Figs 5-8. Abscissa: ratio; ordimte:
no. of particles. I?~?. 5. Analysis of large core-containing refractive bodies with beam positioned on edge. Histogram of the ratios of atomic percents of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c), magnesium and calcium to phosphorus (d).
Since the heights of the peaks are only roughly proportional to the concentrations of each element and cannot be compared to each other without correction for differential absorption, stopping power and fluorescence [l], no scale for the abscissa is presented. It is significant that the X-ray profiles have sharp edges, and that the X-ray profiles are congruent with the SC profile. In electron probe analysis, this provides internal evidence that the constituents of the granules did not diffuse into the cytoplasm during preparation [5]. All bodies contained, in addition to magnesium and calcium, potassium, phosphorus and carbon. The phosphorus and at least some of the carbon can be attributed to the phospholipid component of the granule. Some granules contained sodium and/or chlorine. Sulfur, iron, zinc and copper were not de-
tected, confirming previous reports of Heller & Kopac [7]. Quantitative analysis was carried out according to the procedure previously described [5]. The large core-containing granules were analyzed in two places by positioning the beam first on the light cortex and then on the darker central core. The results of analysis were grouped according to the three morphological types of bodies described above, and are presented in table 1. Since sodium and chlorine occurred only occasionally, they are not included in the table. The remainder of the contents are mostly the carbon and oxygen (of phosphate) associated with the lipid constituents. The standard deviations of each mean are quite large and give some indication of the extent of variation in composition that occurs among individual bodies. This variation is similar in amount to the variation that
7-7
Exptl
Cell Res 76 (1973)
1
Fig. 6. Analysis of large core-containing refractive bodies with beam positioned on core. Histogram of the ratios of atomic percents of calcium to phosphorus (a), magnesium . . to phosphorus ,~ 1. (b), potassium ^ . . to phosphorus (c) and the sum ot calcium, magnesium and potassium to phosphorus (4.
Amoeba proteus refractiue bodies
31
d MEAN
@Ii: 9x
030
040
c
Fin. 7. Analysis of intermediate sized, solid. refractive bodies. Histogram of ratios of atomic percents of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c) and the sum of calcium, magnesium and potassium to phosphorus (d).
0682'-0047
‘IlC
ii20
050
060
occurs among individual cytoplasmic ‘refractile granules’ in T. pyriformis. Similar to the situation in T. pyriformis, when the atomic percent of each of these elements is referred to the phosphorus content, the results are much more uniform [5]. This can be seen in table 2, where the ratios of the atomic percent of each element to the atomic percent of phosphorus are presented. It is evident that the standard deviations are substantially smaller. The mean Ca/P ratio for each group of bodies is similar, as is the mean Mg/P and K/P ratios. It is of interest that the sum of atomic percents for calcium and magnesium as well as calcium, magnesium and potassium in each body are also related
’
VEaY.OZ5G
I
f 00%
1 MEAN-C132 I
ZOO57
!J.EAl\r
t-l
to the amount of phosphorus. This can be seen in the last two lines of table 2. The extent of variation in the ratios of these elements to phosphorus can be seen in figs 5-8. In each of these, the ratios of atomic percents of calcium to phosphorus (5a, 6a, 7a, 8a), magnesium to phosphorus (5b, 6b, 7b, Sb). potassium to phosphorus (5c, 6c, 7c, 8c) and the sum of calcium, magnesium and potassium to phosphorus (54 64 74 8 A) are presented. Since the precision of analysis is about + 2 96, this variability reflects a real variation in composition that occurs among individual granules. As with T. pyriformis, the variation within a cell was less than that among different ceils.
f GO50
d 1 MEAN.0250
G632 t 0041
Fig. 8. Analysis of small, solid, refractive bodies. Histogram of ratios of atomic percent of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c) and the sum of calcium, magnesium and potassium to phosphorus (4.
J
l(J -I
I 5j
01
L--
Cl0
G2G 030 0.40 0
Cl0
C 20 0.50 C60
3.70 Exptl Cell Res 76 (1973)
38 J. R. Coleman et al. Fig. 5, analysis of the edge of the large, core-containing bodies shows that all determinations of the Ca/P ratios fall between narrow limits with a range of 0.35. The K/P ratios have a broad peak and a range of 0.25. The ratios of the sum of calcium, magnesium and potassium to phosphorus in each refractive body are also rather uniform. The values have a broad peak with a range of 0.30. Fig. 6, analysis in the center of the same large core-containing bodies shows that the mean value for the Ca/P ratio is not significantly different from that on the edge, and the range of ratios is 0.30. The K/P ratios from the center of the bodies are not significantly different from those found on the edge, although the range of the distribution, 0.20, falls within slightly narrower limits. The Mg/P ratios on center and edge are not significantly different and fall within a similar range, 0.25. The ratio of the sum of calcium, magnesium and potassium to phosphorus in individual bodies is almost identical in centre and edge, although the range of the distribution is in the center slightly greater, 0.35. Fig. 7 shows the same ratios found in the homogeneous, intermediate-sized bodies. The Mg/P ratios exhibit a sharp peak, and the K/P ratios a broader peak. The range of Ca/P ratios, 0.20, is less than in the larger bodies, while the range of K/P, 0.25, and Mg/P, 0.20, ratios are similar or just slightly less than those of the large bodies. Fig. 8 shows that the Ca/P ratio of the smallest bodies was slightly lower than that of the other bodies and the range of the distribution, 0.25, was similar to that of the other bodies. The K/P ratios did not exhibit a sharp peak, and their distribution and mean were not significantly different than that in the other bodies. The mean Mg/P ratio of the small granules was similar to that of the other bodies and the distribution of values fell within a range similar to those of the Expil
Cell Res 76 (1973)
other size class bodies. Finally, the mean ratio of the sum of calcium, magnesium and potassium to phosphorus exhibits a sharp peak and is similar to that found in the larger bodies, although the distribution, 0.20, of values is somewhat narrower. DISCUSSION The analyses presented here indicate that the refractive bodies vary considerably in their composition but tend to have uniform proportions of calcium, magnesium, potassium and phosphorus. The reasons for the variability in composition are not obvious, but may reflect the availability of materials from the diet at the time the body is forming, The uniformity of metal to phosphorus proportions among all classes of bodies, even the smallest, which are not usually classified as refractive bodies, was unexpected. Heller & Kopac [7] had reported an apparent higher concentration of calcium and magnesium in the core of the largest granules. By electron probe analysis, core and cortex did not differ significantly in the concentrations of all the elements measured. This may be due to the use of conventional aqueous preparative methods by Heller & Kopac. Such methods have been shown to be capable of extracting and redistributing elements as soluble and diffusible as calcium 16). Heller & Kopac 171, using staining methods, found no indication that iron, zinc or copper were present, and this was true for electron probe analysis. The sensitivity of the staining methods employed is not established, but elements present in concentrations less than 0.01 % by weight would probably not be detected by electron probe analysis [I]. The relationship of calcium, magnesium and potassium to phosphorus is not unexpected, since the phosphorus content of the bodies is probably due to the phospholipids
Amoeba proteus refractive bodies they contain. Phospholipids readily bind metallic cations, and in the case of calcium, the formation constant has been reported to be as high as lo* to lo6 [3]. Thus, any metallic cations present in the cytoplasm would be expected to be associated with phospholipids in an equilibrium governed by the formation constant of the metal and phospholipid. The lipid nature of the refractive bodies as well as their content of magnesium and potassium distinguishes them from the crystalline calcium deposits that occur in several species of Spirostromum as reviewed by Pautard [IO]. In these organisms, calcium and small phosphate, as hydroxyapatite, amounts of calcium carbonate, as calcite, occur within membrane-bounded vesicles. These deposits are thought to provide a rigid internal support useful in the burrowing habitat of the organism. However, the composition of the refractile bodies does bear a striking resemblance to the ‘refractive granules’ of Tetrahymena pyriformis. In T. pyriformis, these lipid-containing organelles are composed of the same elements, and the amounts of calcium, magnesium and potassium are a function of the phosphorus content. Because of this, it was suggested that the cytoplasmic concentrations of calcium, magnesium and potassium would be influenced by the equilibrium between the free and phospholipid bound forms of the elements. Thus, the granules might be a ‘buffer’ pool capable of sequestering and releasing these elements with little or no energy expenditure. Such a mechanism could also be an advantage to an organism with the characteristics and habitat of A. proteus. This organism is a free-living, fresh water protozoan, whose only known source of minerals is from engulfed food organisms, or medium ingested through pinocytosis [4]. Thus, minerals tend to appear in the cytoplasm as ‘pulses’. In order to control cyto-
39
plasmic concentrations and maintain them at constant levels, the minerals must be extruded or sequestered. If extruded, they are lost to the organism, a possible disadvantage for an organism which lives in a mineral-poor environment. Active transport of calcium into mitochondria for sequestration also entails disadvantages. First, oxidative phosphorylation is uncoupled until the cytoplasmic level of calcium is returned to its usual low value requires and second, calcium transport substantial expenditure of energy in terms of electron transport [8]. However, the use of phospholipid to sequester these minerals can eliminate these drawbacks. If the minerals were in association with phospholipid then the concentration free in the cytoplasm would be largely a function of the formation constant for each element and the type of phospholipid present. Thus, the movement of an element from free to sequestered form would be governed by mass action considerations and would require little or no expenditure of metabolic energy. The authors thank Mrs Patricia Moran for her skilled technical assistance. The authors are indebted to Dr Cicily Chapman-Andresen for her advice and counsel. Jytte R. Nilsson wishes to thank James R. Coleman most sincerely for excellent and stimulating working conditions during her stay at the University of Rochester. Furthermore, she gratefully acknowledges the financial support of the Carl&erg Foundation, Copenhagen. This report is based in part on work performed under contract with the Atomic Enertzv Commission at the University of Rochester Atom% Energy Project and assigned Report Number UR-3490-110 and in part on work supported by USPHS Research Grant AM14272.
REFERENCES Andersen, C A, Methods of biochemical analysis (ed D Glick) vol. 15, p. 147. Interscience, New York (1967). Byrne, J M, Quart j micr sci 104 (1963) 44.5. Carr, C W & Chang, K Y, Cellular mechanism for calcium transfer and homeostasis (ed G V Nichols & R H Wasserman) p. 41. Academic Press, New York (1971). Chapman-Andresen, C, Ann rev microbial 25 (1971) 27. Exptl Cell Res 76 (1973)
40 J. R. Coleman et al. 5. Coleman, J R, Nilsson, J R, Warner, R R & Batt, P, Exptl cell res 74 (1972) 207. 6. Coleman, J R & Terepka, A R, J histochem cytothem 20 (1972) 401. 7. Heller, I M & Kopac, M J, Exptl cell res 11 (1956)
10. Pautard, F G E, Biological calcification, cellular and molecular aspects (ed H Schraer) p. 105. Appleton-Century-Crofts, New York (1970). 11. Warner, R R, Proc VI natl electron probe analysis (ed L Vassamillet) p. 42A. Pittsburgh (1970).
206.
8. Lehninger, A L, Biochem j 119 (1970) 129. 9. Mast, S 0 & Doyle, W L, Arch protistenk 86 (1935) 279.
Exptl
Cell Res 76 (1973)
Received May 9, 1972 Revised version received August 7, 1972
Experimental Cell Research 76 (1973) 31-40
ELECTRON
PROBE ANALYSIS IN AMOEBA
J. R. COLEMAN,
OF REFRACTIVE
BODIES
PROTEUS
JYTTE R. NILSSON, R. R. WARNER and P. BATT
Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642, USA and Biological Institute of the Carlsberg Foundation, Copenhagen, Denmark
SUMMARY Phospholipid organelles, termed refractive bodies, occur in the cytoplasm of Amoeba proteus. Electron probe analysis of individual granules in situ shows that their composition is variable, and that they are rich in calcium, magnesium, and potassium. The amount of calcium,magnesium, and potassium, both singly and in sum, is proportional to the phosphorus content. This is consistent with the view that the metals are complexed with a phospholipid component of the refractive body. These observations indicate that the refractive bodies of Amoeba proteus are homologous with the refractile granules of Tetrahymena pyriformis, and suggest that the two may play a similar role in the regulation of intracellular metal concentration.
Recently, electron probe analysis of lipid ‘refractile granules’ in the cytoplasm of Tetrahymena pyriformis indicated that they were intracellular stores of calcium, magnesium and potassium [5]. Quantitative electron probe analysis revealed that the amount of each of these elements in a refractile granule was proportional to the phosphorus content of the granule, suggesting that the metal ions were complexed with a phospholipid component [5]. It was pointed out that the equilibrium between the free and lipid bound forms of each of the ions would help determine the concentration of each ion free in the cytoplasm. Thus, the granules would serve as intracellular ‘buffers’ for these metabolically important ions. The particular advantages of such a mechanism for the intracellular regulation of calcium, an ion used to control several cell processes, were discussed with regard to the habitat and phy3-
721815
siology of T. pyriformis [5]. However, it was not known whether these organelles were restricted to just this one species or whether they could be a feature common to other protozoa. Morphologically similar organelles, termed refractive bodies, occur in the cytoplasm of Amoeba proteus [9]. Histochemical investigations showed that they are phospholipid and contain inorganic materials identified as calcium and magnesium [2, 71. This composition is superficially similar to that of the T. pyriformis granules, but it is not sufficient to establish an homology between the two organelles. Consequently, a quantitative electron probe analysis of individual refractive bodies in situ was performed to determine (a) whether the bodies of A. proteus have a composition similar to the granules of T. pyriformis, and (b) whether the amount of calcium, magnesium and potassium contained Exptl Cell Res 76 (1973)
32
J. R. Coleman et al. in the refractive bodies is proportional to the phosphorus content, as in T. pyrifoumis. The use of the electron probe eliminated the hazards of redistribution and loss associated with the isolation of organelles containing readily soluble constituents, and analysisIof individual granules permitted the detection of variations among individuals and size classes of organelles. MATERIALS
AND METHODS
Amoeba proteus, obtained from Wards Scientific, Rochester, N.Y., were cultured in the medium supplied and prepared for analysis the day after arrival. Single cells were placed on a clean, silicon disc in large drops of medium and permitted to attach to the substrate. Excess liquid was quickly removed with filter paper and cells were heat-fixed by passing the silicon disc bearing the cells through the flame of a propane torch, essentially the same procedure used previously for T. pyriformis [5]. Morphological preservation was assessedwith Leitz reflecting optics at 500x and 1 000 x magnification. Comparison of sample current and X-ray profiles was used to insure that no detectable redistribution of the elements of interest had occurred during preparation [5]. Electron probe analysis was performed on 79 granules in 5 cells from 3 different cultures according to techniques described previously [5, 61. Granules were analyzed for C, Na, K, Mg, Ca, S, Fe, Cu, Zn, Cl and P. Quantitative analysis was performed as before, using the BICEP computer program on an IBM 360/44 to correct X-ray intensities [ll]. This procedure is accurate to about il0 sb for atomic ratios and replicate measurements indicate precision is about +2 % [5].
RESULTS
Fig. 1. Sample current (SC) and X-ray images of Amoeba proteus. The sample current image (a) shows
the outline of the dried cell containing numerous bright refractive bodies mainly localized in tie cell body. The calcium Kcc X-ray image (b) shows that the largest part of the total cell calcium is contained within the refractile bodies. The grid marks cover identical areas in each image and the source of any X-ray signal can be identified in the SC image by referring to the grid. The phosphorus X-ray image (c) Expll
Cell Res 76 (1973)
The sample current (SC) image of a heat fixed Amoeba proteus appears in fig. 1. The refractive bodies appear as bright particles throughout the central main portion of the cytoplasm and also in the pseudopodia. About 800 refractile bodies are present in this cell. Other cell organelles are not distinct in the SC image because contrast is a function of local atomic number differences, and most shows that phosphorus is distributed throughout the cell, and that the refractive bodies are especially rich in phosphorus. Each grid division represents 40 ,um.
Amoeba proteus refractive bodies
33
Fig. 2. Sample current and X-ray images of refractive bodies. (a) SC image at t ,rm/screen division. Large
‘core’ containing bodies as well as smaller ones are visible. The smallest bodies are only faintly visible in the background. (b) a higher magnification, 1 pm/screen division, two large core-containing bodies are visible. A smaller adjacent body is indistinct in this SC image but appears clearly in the X-ray images; (c) is the calcium Kcc X-ray image of the bodies seen in (a); (b) showing that calcium is present in the granules at a higher concentration than in the surrounding cytoplasm; (d) phosphorus Ka X-ray image of the bodies in (b), showing a concentration of phosphorus in the bodies; (e) potassium Kcc X-ray image; (f) the magnesium Ka X-ray image of the same bodies, showing that the granules contain a greater concentration of each of these elements than the surrounding cytoplasm. Exptl Cell Res 76 (1973)
34
J. R. Coleman et al.
PHOSPHORUS
Fig. 3. Sample current image of refractive body at
0.5 /&m/screen division. The SC and X-ray profiles in fig. 4 were generated by driving the beam across this granule along a path indicated by the grid line marked by white triangles.
of the cell is composed of elements with similar atomic numbers. A major proportion of the total cell calcium is contained in the refractive bodies, as can be seen in the calcium X-ray image (fig. 1b). The phosphorus X-ray image (fig. 1 c) shows that while the bodies are rich in phosphorus, the surrounding cytoplasm also contains a substantial amount of this element. At higher magnifications, morphological characteristics of individual bodies can be resolved. In fig. 2a, the various types of granules can be distinguished: (1) large bodies about 1.44 pm or more in diameter, showing a central darker ‘core’; (2) intermediate spherical bodies about 1.6 pm in diameter, with no core and (3) small bodies about 0.8 pm in diameter, only faintly visible in the background at this magnification. These smallest bodies are not usually classified as refractive bodies. The solid and the coreExptl
Cell Res 76 (1973)
SAMPLECURRENT
Fig. 4. Sample current (botlom) profile of body seen
in SC image in fig. 3. The potassium Ka X-ray profile, calcium Kcc X-ray profile and phosphorus Ka X-ray profile are seen above and congruent with the SC profile.
Amoeba proteus refractive bodies 35 Table 1. Means of atomic percent for analysis of several elements in refractile granules of Amoeba proteus Figures within
parentheses are S.D. Large Edge (N=26)
Ca Mg K P
4.19 1.54 2.85 12.78
Intermediate (N=28)
Center (N= 21)
(1.29) (0.88) (1.01) (3.14)
3.23 0.75 1.48 8.54
4.04 1.09 2.76 11.63
(1.19) (0.40) (0.46) (2.51)
(1.06) (0.33) (0.78) (1.86)
Small (N=25) 3.80 1.86 3.58 14.72
(1.49) (0.61) (0.86) (3.38)
-
containing bodies are spherical in vivo, but it is not possible to determine whether they maintain this shape or whether they become discoid in the fixed cells. The three-dimensional shape of the bodies, however, does not influence the results of analysis when atomic ratios are employed. Fig. 2b shows the sample current of two large adjacent bodies. An intermediate sized body is alongside but indistinct in this mode of display. The calcium (2c), phosphorus (2d), potassium (2e), and magnesium (2f) X-ray images are presented in the samefigure. It can be seen in these images that the concentrations of each of these elements in the bodies is greater than in the surrounding cytoplasm, and that the localization of each element corresponds quite closely to the shapes of the bodies as seen in the SC image.
This is readily perceived in a comparison of SC and X-ray profiles. Fig. 3 shows the SC image of a single large core-containing body. Profiles were generatedby driving the electron beam at a constant rate in a straight line over the same path across the body. The resulting sample current or characteristic X-ray intensities were recorded on successivesweeps of the beam. These can be seen in fig. 4. The lowest trace in fig. 4 is the SC profile from the body in fig. 3 and corresponds to a trace of density across the horizontal grid line visible near the center of the image. The potassium X-ray profile in fig. 4 is directly above the SC profile and its shape is congruent with the SC profile. Above this is the calcium profile, also congruent with the SC profile. The topmost trace is the phosphorus X-ray profile, also congruent with the SC profile.
Table 2. Mean ratios of metals to phosphorus (atomic percent) in cytoplasmic granules in Amoeba proteus Figures within
parentheses are S.D. Large Edge (N-26)
Ca/P MU K/P Ca + Mg/P Ca +Mg+K P
0.332 0.119 0.225 0.450
Center (N=21) (0.074) (0.054) (0.058) (0.054)
0.676 (0.065)
0.375 0.096 0.177 0.471
(0.065) (0.060) (0.041) (0.051)
0.647 (0.072)
Intermediate (N = 28)
Small (N-25)
0.348 0.095 0.234 0.448
0.250 0.132 0.250 0.383
(0.013) (0.033) (0.046) (0.018)
0.682 (0.047)
(0.054) (0.050) (0.057) (9.038)
0.632 (0.041)
Exptl Cell Res 76 (1973)
36 J. R. Coleman et al.
Figs 5-8. Abscissa: ratio; ordimte:
no. of particles. I?~?. 5. Analysis of large core-containing refractive bodies with beam positioned on edge. Histogram of the ratios of atomic percents of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c), magnesium and calcium to phosphorus (d).
Since the heights of the peaks are only roughly proportional to the concentrations of each element and cannot be compared to each other without correction for differential absorption, stopping power and fluorescence [l], no scale for the abscissa is presented. It is significant that the X-ray profiles have sharp edges, and that the X-ray profiles are congruent with the SC profile. In electron probe analysis, this provides internal evidence that the constituents of the granules did not diffuse into the cytoplasm during preparation [5]. All bodies contained, in addition to magnesium and calcium, potassium, phosphorus and carbon. The phosphorus and at least some of the carbon can be attributed to the phospholipid component of the granule. Some granules contained sodium and/or chlorine. Sulfur, iron, zinc and copper were not de-
tected, confirming previous reports of Heller & Kopac [7]. Quantitative analysis was carried out according to the procedure previously described [5]. The large core-containing granules were analyzed in two places by positioning the beam first on the light cortex and then on the darker central core. The results of analysis were grouped according to the three morphological types of bodies described above, and are presented in table 1. Since sodium and chlorine occurred only occasionally, they are not included in the table. The remainder of the contents are mostly the carbon and oxygen (of phosphate) associated with the lipid constituents. The standard deviations of each mean are quite large and give some indication of the extent of variation in composition that occurs among individual bodies. This variation is similar in amount to the variation that
7-7
Exptl
Cell Res 76 (1973)
1
Fig. 6. Analysis of large core-containing refractive bodies with beam positioned on core. Histogram of the ratios of atomic percents of calcium to phosphorus (a), magnesium . . to phosphorus ,~ 1. (b), potassium ^ . . to phosphorus (c) and the sum ot calcium, magnesium and potassium to phosphorus (4.
Amoeba proteus refractiue bodies
31
d MEAN
@Ii: 9x
030
040
c
Fin. 7. Analysis of intermediate sized, solid. refractive bodies. Histogram of ratios of atomic percents of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c) and the sum of calcium, magnesium and potassium to phosphorus (d).
0682'-0047
‘IlC
ii20
050
060
occurs among individual cytoplasmic ‘refractile granules’ in T. pyriformis. Similar to the situation in T. pyriformis, when the atomic percent of each of these elements is referred to the phosphorus content, the results are much more uniform [5]. This can be seen in table 2, where the ratios of the atomic percent of each element to the atomic percent of phosphorus are presented. It is evident that the standard deviations are substantially smaller. The mean Ca/P ratio for each group of bodies is similar, as is the mean Mg/P and K/P ratios. It is of interest that the sum of atomic percents for calcium and magnesium as well as calcium, magnesium and potassium in each body are also related
’
VEaY.OZ5G
I
f 00%
1 MEAN-C132 I
ZOO57
!J.EAl\r
t-l
to the amount of phosphorus. This can be seen in the last two lines of table 2. The extent of variation in the ratios of these elements to phosphorus can be seen in figs 5-8. In each of these, the ratios of atomic percents of calcium to phosphorus (5a, 6a, 7a, 8a), magnesium to phosphorus (5b, 6b, 7b, Sb). potassium to phosphorus (5c, 6c, 7c, 8c) and the sum of calcium, magnesium and potassium to phosphorus (54 64 74 8 A) are presented. Since the precision of analysis is about + 2 96, this variability reflects a real variation in composition that occurs among individual granules. As with T. pyriformis, the variation within a cell was less than that among different ceils.
f GO50
d 1 MEAN.0250
G632 t 0041
Fig. 8. Analysis of small, solid, refractive bodies. Histogram of ratios of atomic percent of calcium to phosphorus (a), magnesium to phosphorus (b), potassium to phosphorus (c) and the sum of calcium, magnesium and potassium to phosphorus (4.
J
l(J -I
I 5j
01
L--
Cl0
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Cl0
C 20 0.50 C60
3.70 Exptl Cell Res 76 (1973)
38 J. R. Coleman et al. Fig. 5, analysis of the edge of the large, core-containing bodies shows that all determinations of the Ca/P ratios fall between narrow limits with a range of 0.35. The K/P ratios have a broad peak and a range of 0.25. The ratios of the sum of calcium, magnesium and potassium to phosphorus in each refractive body are also rather uniform. The values have a broad peak with a range of 0.30. Fig. 6, analysis in the center of the same large core-containing bodies shows that the mean value for the Ca/P ratio is not significantly different from that on the edge, and the range of ratios is 0.30. The K/P ratios from the center of the bodies are not significantly different from those found on the edge, although the range of the distribution, 0.20, falls within slightly narrower limits. The Mg/P ratios on center and edge are not significantly different and fall within a similar range, 0.25. The ratio of the sum of calcium, magnesium and potassium to phosphorus in individual bodies is almost identical in centre and edge, although the range of the distribution is in the center slightly greater, 0.35. Fig. 7 shows the same ratios found in the homogeneous, intermediate-sized bodies. The Mg/P ratios exhibit a sharp peak, and the K/P ratios a broader peak. The range of Ca/P ratios, 0.20, is less than in the larger bodies, while the range of K/P, 0.25, and Mg/P, 0.20, ratios are similar or just slightly less than those of the large bodies. Fig. 8 shows that the Ca/P ratio of the smallest bodies was slightly lower than that of the other bodies and the range of the distribution, 0.25, was similar to that of the other bodies. The K/P ratios did not exhibit a sharp peak, and their distribution and mean were not significantly different than that in the other bodies. The mean Mg/P ratio of the small granules was similar to that of the other bodies and the distribution of values fell within a range similar to those of the Expil
Cell Res 76 (1973)
other size class bodies. Finally, the mean ratio of the sum of calcium, magnesium and potassium to phosphorus exhibits a sharp peak and is similar to that found in the larger bodies, although the distribution, 0.20, of values is somewhat narrower. DISCUSSION The analyses presented here indicate that the refractive bodies vary considerably in their composition but tend to have uniform proportions of calcium, magnesium, potassium and phosphorus. The reasons for the variability in composition are not obvious, but may reflect the availability of materials from the diet at the time the body is forming, The uniformity of metal to phosphorus proportions among all classes of bodies, even the smallest, which are not usually classified as refractive bodies, was unexpected. Heller & Kopac [7] had reported an apparent higher concentration of calcium and magnesium in the core of the largest granules. By electron probe analysis, core and cortex did not differ significantly in the concentrations of all the elements measured. This may be due to the use of conventional aqueous preparative methods by Heller & Kopac. Such methods have been shown to be capable of extracting and redistributing elements as soluble and diffusible as calcium 16). Heller & Kopac 171, using staining methods, found no indication that iron, zinc or copper were present, and this was true for electron probe analysis. The sensitivity of the staining methods employed is not established, but elements present in concentrations less than 0.01 % by weight would probably not be detected by electron probe analysis [I]. The relationship of calcium, magnesium and potassium to phosphorus is not unexpected, since the phosphorus content of the bodies is probably due to the phospholipids
Amoeba proteus refractive bodies they contain. Phospholipids readily bind metallic cations, and in the case of calcium, the formation constant has been reported to be as high as lo* to lo6 [3]. Thus, any metallic cations present in the cytoplasm would be expected to be associated with phospholipids in an equilibrium governed by the formation constant of the metal and phospholipid. The lipid nature of the refractive bodies as well as their content of magnesium and potassium distinguishes them from the crystalline calcium deposits that occur in several species of Spirostromum as reviewed by Pautard [IO]. In these organisms, calcium and small phosphate, as hydroxyapatite, amounts of calcium carbonate, as calcite, occur within membrane-bounded vesicles. These deposits are thought to provide a rigid internal support useful in the burrowing habitat of the organism. However, the composition of the refractile bodies does bear a striking resemblance to the ‘refractive granules’ of Tetrahymena pyriformis. In T. pyriformis, these lipid-containing organelles are composed of the same elements, and the amounts of calcium, magnesium and potassium are a function of the phosphorus content. Because of this, it was suggested that the cytoplasmic concentrations of calcium, magnesium and potassium would be influenced by the equilibrium between the free and phospholipid bound forms of the elements. Thus, the granules might be a ‘buffer’ pool capable of sequestering and releasing these elements with little or no energy expenditure. Such a mechanism could also be an advantage to an organism with the characteristics and habitat of A. proteus. This organism is a free-living, fresh water protozoan, whose only known source of minerals is from engulfed food organisms, or medium ingested through pinocytosis [4]. Thus, minerals tend to appear in the cytoplasm as ‘pulses’. In order to control cyto-
39
plasmic concentrations and maintain them at constant levels, the minerals must be extruded or sequestered. If extruded, they are lost to the organism, a possible disadvantage for an organism which lives in a mineral-poor environment. Active transport of calcium into mitochondria for sequestration also entails disadvantages. First, oxidative phosphorylation is uncoupled until the cytoplasmic level of calcium is returned to its usual low value requires and second, calcium transport substantial expenditure of energy in terms of electron transport [8]. However, the use of phospholipid to sequester these minerals can eliminate these drawbacks. If the minerals were in association with phospholipid then the concentration free in the cytoplasm would be largely a function of the formation constant for each element and the type of phospholipid present. Thus, the movement of an element from free to sequestered form would be governed by mass action considerations and would require little or no expenditure of metabolic energy. The authors thank Mrs Patricia Moran for her skilled technical assistance. The authors are indebted to Dr Cicily Chapman-Andresen for her advice and counsel. Jytte R. Nilsson wishes to thank James R. Coleman most sincerely for excellent and stimulating working conditions during her stay at the University of Rochester. Furthermore, she gratefully acknowledges the financial support of the Carl&erg Foundation, Copenhagen. This report is based in part on work performed under contract with the Atomic Enertzv Commission at the University of Rochester Atom% Energy Project and assigned Report Number UR-3490-110 and in part on work supported by USPHS Research Grant AM14272.
REFERENCES Andersen, C A, Methods of biochemical analysis (ed D Glick) vol. 15, p. 147. Interscience, New York (1967). Byrne, J M, Quart j micr sci 104 (1963) 44.5. Carr, C W & Chang, K Y, Cellular mechanism for calcium transfer and homeostasis (ed G V Nichols & R H Wasserman) p. 41. Academic Press, New York (1971). Chapman-Andresen, C, Ann rev microbial 25 (1971) 27. Exptl Cell Res 76 (1973)
40 J. R. Coleman et al. 5. Coleman, J R, Nilsson, J R, Warner, R R & Batt, P, Exptl cell res 74 (1972) 207. 6. Coleman, J R & Terepka, A R, J histochem cytothem 20 (1972) 401. 7. Heller, I M & Kopac, M J, Exptl cell res 11 (1956)
10. Pautard, F G E, Biological calcification, cellular and molecular aspects (ed H Schraer) p. 105. Appleton-Century-Crofts, New York (1970). 11. Warner, R R, Proc VI natl electron probe analysis (ed L Vassamillet) p. 42A. Pittsburgh (1970).
206.
8. Lehninger, A L, Biochem j 119 (1970) 129. 9. Mast, S 0 & Doyle, W L, Arch protistenk 86 (1935) 279.
Exptl
Cell Res 76 (1973)
Received May 9, 1972 Revised version received August 7, 1972