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Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus
456
Preliminary notes
This work was supported by Research Grants (nos. 754126 and 734044) from the Ministry of Education of Japan.
References 1. Nickel], L G & Torrey, J G, Science 166 (1969) 1068. 2. Power, J B, Cummins, S E & Cocking, E, Nature 225 (1970) 1016. 3. Potrykus, I, Nature new biol 231 (1971) 57. 4. Ito, M, Bot mag 86 (1973) 133. 5. Erickson, R 0, Am j bot 35 (1948) 729. 6. Sparrow, A H & Sparrow, R C, Stain tech 24 (1949) 47. 7. Ito, M & Stern, H, Dev biol 16 (1967) 36. 8. White, P R, The cultivation of animal and plant cells, 2nd edn. Ronald Press, New York (1963). 9. Carlson, P S, Smith, H H & Dearing, R D, Proc natl acad sci US 69 (1972) 2292. Received February 1, 1973 Revised version received March 15, 1973
Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus K. BRAATZ-SCHADE, M. HABEREY and W. STOCKEM, Institute of Cytology and Micromorphology, University
of Bonn, 53 Bonn I, BRD
Summary. The various motile activities and cell shapes of Amoeba proteus grown in Chalkley’s solution are
correlated with definitive electrical membrane potentials. The same correlations were found when definitive motile activities and cell shapes were experimentally induced by changing the pH of the culture medium. The highest values of membrane potential (-70 mV) were measured in monopodial amebae during active locomotion. In resting cells, which prevail in acid or basic media, the membrane potential decreases to - 5 mV. In those resting cells, which also stop internal cytoplasmic movement at basic pH, the membrane potential turns positive ( + 9 mV - + 30 mV).
Several investigators [l, 2, 3, 51 have shown local differences to occur in the membrane potential along locomoting amebae. In addition, Jeon & Bell [12] demonstrated that some of the factors which influence movements in Amoeba proteus simultaneously modify the value of its membrane potential. In previous work from this laboratory [6] it was found that changes in pH of the medium affect the Exptl
Cell Res 80 (1973)
morphology and locomotion in Amoeba proteus. The aim of the experiments presented here is, therefore, to examine the correlation between the electrical potentials, motile activity and cell shape in this ameba, as influenced by the pH of the medium as well as under physiological conditions. Materials and Methods Amoeba proteus (dark strain originated from Princeton
collection) was cultured in Chalkley’s medium [lo] and, after washing, was transferred to media of various pH values as described by Braatz-Schade & Stockem [6]. To avoid calcium precipitation in basic solutions the media of pH 7.0 to 9.5 were buffered with constant ionic strength Tris-NC1 buffer. Measurements of the membrane potentials were carried out by the method described by Bingley [4]. Glass microcapillary Ag/AgCl electrodes filled with 3 M KC1 solution were coupled with a high input resistance amplifier connected to a Beckmann automatic recorder. The measuring electrode was always inserted into the middle-endoplasmic region of the amebae.
Results As was found by Braatz-Schade & Stockem [6], at any given pH value of the medium, different morphological forms of Amoeba proteus can be observed (cf fig. l), but one of them always dominates. The first series of measurements was carried out to determine the value of the membrane potential in these characteristic forms of amebae. The results of these measurements are given in fig. 2. They show that the highest values of the negative membrane potential (-70 mV) are found in amebae at pH 5.0, i.e. in actively locomoting monopodial specimens. In more acid or basic media the membrane potential of amebae decreases to - 5 and - 15 mV at pH 2.0 and 9.5 respectively. In general it was observed that non-locomoting amebae (forms: a, b, f, g, h) show lower negative membrane potentials than amebae which actively locomote (c, d and e forms). In some cases in basic media, positive values of the membrane potential were recorded in rounded
Preliminary notes 451
Fig. 1. Dominant forms of A. proteus found in media of various pH: form a, pH 2.0; b, pH 4.0; c, pH 5.0; d, pH 6.0; e, pH 6.5;f, pH 7.5; g, pH 8.5; h, pH 9.5
(cf t61) Fig. 3. Abscissa: form of amebae; ordinate: PD [mV].
cells in which no cytoplasmic currents could Average membrane potentials and SD. in the same forms of A. proteus as in fig. 2, but measured in be observed (cf figs 2, 3, f’, g’, h’). medium of pH 6.5 (cf text). All of these dominating forms of amebae characteristic for a given pH of the medium can also be found sporadically in physiologi- cal media [6]. The second series of measurements was thus carried out on amebae in medium of pH 6.5 but on chosen specimensof Amoeba proteus denoted as a-h forms. Results of these measurements shown in fig. 3 correspond almost exactly to the results of the first series of measurements. This shows that the membrane potential value in Amoeba proteus is primarily correlated with the morphological form and motile activity of the cell. The average membrane potential for a statistical inhomogenous sample of amebae at any particular pH would be represented by the sum of products of the average potential of defined cell types a-h and their frequencies, i.e.
Fig. 2. Abscissa: pH value; ordinate: PD [mV]. Average membrane potentials and S.D. in dominant forms of A. proteus as measured in media of various pH (cf fig. 1). Forms f’, g’ and h’ are morphologically identical with forms f, g and h respectively, but they do not show cytoplasmic currents (cf text). 30-731804
1 PD, 2 I’D, + 3 L+ . . . + !.!!f PD,H=n,Lm nb m m n,
+A, X nh
Exptl Cell Res 80 (1973)
458
Preliminary notes
where: m=n,+n,+... +n, (number of all amebae in the sample), na, nb .. . nh (number of amebae of the forms a, b, .. . h, respectively) PD,, PDb . .. PDI, (potential differences found in individual cells of the forms a, b, ... h). The differences among average membrane potential values thus calculated in amebae at various pH therefore result from the fact that the occurrence of particular cell forms depends on the pH of the medium, i.e. 2 =f(pH),
2 =f(pH)
etc.
whereas average membrane potentials for any form of ameba, i.e. ZPD, m=1na
3 PD, , PD,=kb-etc.
are constant, and independent of the pH of the medium.
the plasma membrane in erythrocytes which were induced by changes in the pH of the medium. Membrane structure and permeability and consequently the distribution of ions between the cytoplasm and external medium, which is what is measured in microelectrode experiments, can also be modified by numerous external agents. Among them can be mentioned changes in the Ca2+, NH:, K+ cont. or other ions, lipid-soluble anesthetics, detergents, macromolecules adsorbed onto the cell surface, contacts with solid surfaces, etc. [7, 8, 9, 13, 14, 15 and other authors]. All these factors are known to influence the cell morphology and cellular motility. Studies on the correlation between electrophysiological properties of amebae and their reactions to external factors will be continued since we believe they can give some information on the mechanisms which control cellular shape and movements. The authors wish to thank Dr W. Korohoda for discussion and valuable help in preparing the manuscript. They thank Mr E. Samans for excellent technical assistance.
Discussion The results presented in a previous communication indicated that the concentration of H+ ions in the medium is one of the factors which induce characteristic responses in amebae [6]. These responses were found to be correlated with definite values of the cell membrane potential. Amebae can, however, react similarly to other factors, since various forms of cells can also be observed at physiological pH. The simplest explanation for this observation would be that the external H+-cont. indirectly influences the state and permeability of the cell membrane and the state of the amebic cytoplasm. As not all amebae respond typically to the pH-changes, the effectiveness of this influence seems to depend on the physiological state of the amebae. Heard & Seaman [1 I] showed modifications in the stability of Exptl CeII Res 80 (1973)
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Batueva, 1 V, Cytology 6 (1964) 209. - Ibid 7 (1965) 188. Batueva, I V & Lev, A A, Cytology 9 (1967) 680. Bingley, M S, Exptl cell res 34 (1964) 266. - J exptl biol 45 (1966) 251. Braatz-Schade, K & Stockem, W, Arch Protistenk 114 (1972) 272. Brandt, P W & Freeman, A R, Science 155 (1967) 582. Brewer. J E & Bell. L G E. J cell sci 4 (1969) . , 17. - Ibid 7 (1970) 549. Haberey, M & Stockem, W, Mikrokosmos 60 (1971) 33. Heard. D H & Seaman, G V F, J gen physiol 43 (1960) 653. Jeon, K W & Bell, L G E, Exptl cell res 27 (1962) 350. Korohoda, W, Acta protozool 11 (1972) 333. Korohoda. W. Ambrose, E J & Forrester, J A, Folia biol i5 (i967) 371. Wolpert, L & Gingell, D, Symp sot exptl biol 22 (1968) 169.
Received February 1, 1973 Revised version received May 7, 1973
Preliminary notes
This work was supported by Research Grants (nos. 754126 and 734044) from the Ministry of Education of Japan.
References 1. Nickel], L G & Torrey, J G, Science 166 (1969) 1068. 2. Power, J B, Cummins, S E & Cocking, E, Nature 225 (1970) 1016. 3. Potrykus, I, Nature new biol 231 (1971) 57. 4. Ito, M, Bot mag 86 (1973) 133. 5. Erickson, R 0, Am j bot 35 (1948) 729. 6. Sparrow, A H & Sparrow, R C, Stain tech 24 (1949) 47. 7. Ito, M & Stern, H, Dev biol 16 (1967) 36. 8. White, P R, The cultivation of animal and plant cells, 2nd edn. Ronald Press, New York (1963). 9. Carlson, P S, Smith, H H & Dearing, R D, Proc natl acad sci US 69 (1972) 2292. Received February 1, 1973 Revised version received March 15, 1973
Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus K. BRAATZ-SCHADE, M. HABEREY and W. STOCKEM, Institute of Cytology and Micromorphology, University
of Bonn, 53 Bonn I, BRD
Summary. The various motile activities and cell shapes of Amoeba proteus grown in Chalkley’s solution are
correlated with definitive electrical membrane potentials. The same correlations were found when definitive motile activities and cell shapes were experimentally induced by changing the pH of the culture medium. The highest values of membrane potential (-70 mV) were measured in monopodial amebae during active locomotion. In resting cells, which prevail in acid or basic media, the membrane potential decreases to - 5 mV. In those resting cells, which also stop internal cytoplasmic movement at basic pH, the membrane potential turns positive ( + 9 mV - + 30 mV).
Several investigators [l, 2, 3, 51 have shown local differences to occur in the membrane potential along locomoting amebae. In addition, Jeon & Bell [12] demonstrated that some of the factors which influence movements in Amoeba proteus simultaneously modify the value of its membrane potential. In previous work from this laboratory [6] it was found that changes in pH of the medium affect the Exptl
Cell Res 80 (1973)
morphology and locomotion in Amoeba proteus. The aim of the experiments presented here is, therefore, to examine the correlation between the electrical potentials, motile activity and cell shape in this ameba, as influenced by the pH of the medium as well as under physiological conditions. Materials and Methods Amoeba proteus (dark strain originated from Princeton
collection) was cultured in Chalkley’s medium [lo] and, after washing, was transferred to media of various pH values as described by Braatz-Schade & Stockem [6]. To avoid calcium precipitation in basic solutions the media of pH 7.0 to 9.5 were buffered with constant ionic strength Tris-NC1 buffer. Measurements of the membrane potentials were carried out by the method described by Bingley [4]. Glass microcapillary Ag/AgCl electrodes filled with 3 M KC1 solution were coupled with a high input resistance amplifier connected to a Beckmann automatic recorder. The measuring electrode was always inserted into the middle-endoplasmic region of the amebae.
Results As was found by Braatz-Schade & Stockem [6], at any given pH value of the medium, different morphological forms of Amoeba proteus can be observed (cf fig. l), but one of them always dominates. The first series of measurements was carried out to determine the value of the membrane potential in these characteristic forms of amebae. The results of these measurements are given in fig. 2. They show that the highest values of the negative membrane potential (-70 mV) are found in amebae at pH 5.0, i.e. in actively locomoting monopodial specimens. In more acid or basic media the membrane potential of amebae decreases to - 5 and - 15 mV at pH 2.0 and 9.5 respectively. In general it was observed that non-locomoting amebae (forms: a, b, f, g, h) show lower negative membrane potentials than amebae which actively locomote (c, d and e forms). In some cases in basic media, positive values of the membrane potential were recorded in rounded
Preliminary notes 451
Fig. 1. Dominant forms of A. proteus found in media of various pH: form a, pH 2.0; b, pH 4.0; c, pH 5.0; d, pH 6.0; e, pH 6.5;f, pH 7.5; g, pH 8.5; h, pH 9.5
(cf t61) Fig. 3. Abscissa: form of amebae; ordinate: PD [mV].
cells in which no cytoplasmic currents could Average membrane potentials and SD. in the same forms of A. proteus as in fig. 2, but measured in be observed (cf figs 2, 3, f’, g’, h’). medium of pH 6.5 (cf text). All of these dominating forms of amebae characteristic for a given pH of the medium can also be found sporadically in physiologi- cal media [6]. The second series of measurements was thus carried out on amebae in medium of pH 6.5 but on chosen specimensof Amoeba proteus denoted as a-h forms. Results of these measurements shown in fig. 3 correspond almost exactly to the results of the first series of measurements. This shows that the membrane potential value in Amoeba proteus is primarily correlated with the morphological form and motile activity of the cell. The average membrane potential for a statistical inhomogenous sample of amebae at any particular pH would be represented by the sum of products of the average potential of defined cell types a-h and their frequencies, i.e.
Fig. 2. Abscissa: pH value; ordinate: PD [mV]. Average membrane potentials and S.D. in dominant forms of A. proteus as measured in media of various pH (cf fig. 1). Forms f’, g’ and h’ are morphologically identical with forms f, g and h respectively, but they do not show cytoplasmic currents (cf text). 30-731804
1 PD, 2 I’D, + 3 L+ . . . + !.!!f PD,H=n,Lm nb m m n,
+A, X nh
Exptl Cell Res 80 (1973)
458
Preliminary notes
where: m=n,+n,+... +n, (number of all amebae in the sample), na, nb .. . nh (number of amebae of the forms a, b, .. . h, respectively) PD,, PDb . .. PDI, (potential differences found in individual cells of the forms a, b, ... h). The differences among average membrane potential values thus calculated in amebae at various pH therefore result from the fact that the occurrence of particular cell forms depends on the pH of the medium, i.e. 2 =f(pH),
2 =f(pH)
etc.
whereas average membrane potentials for any form of ameba, i.e. ZPD, m=1na
3 PD, , PD,=kb-etc.
are constant, and independent of the pH of the medium.
the plasma membrane in erythrocytes which were induced by changes in the pH of the medium. Membrane structure and permeability and consequently the distribution of ions between the cytoplasm and external medium, which is what is measured in microelectrode experiments, can also be modified by numerous external agents. Among them can be mentioned changes in the Ca2+, NH:, K+ cont. or other ions, lipid-soluble anesthetics, detergents, macromolecules adsorbed onto the cell surface, contacts with solid surfaces, etc. [7, 8, 9, 13, 14, 15 and other authors]. All these factors are known to influence the cell morphology and cellular motility. Studies on the correlation between electrophysiological properties of amebae and their reactions to external factors will be continued since we believe they can give some information on the mechanisms which control cellular shape and movements. The authors wish to thank Dr W. Korohoda for discussion and valuable help in preparing the manuscript. They thank Mr E. Samans for excellent technical assistance.
Discussion The results presented in a previous communication indicated that the concentration of H+ ions in the medium is one of the factors which induce characteristic responses in amebae [6]. These responses were found to be correlated with definite values of the cell membrane potential. Amebae can, however, react similarly to other factors, since various forms of cells can also be observed at physiological pH. The simplest explanation for this observation would be that the external H+-cont. indirectly influences the state and permeability of the cell membrane and the state of the amebic cytoplasm. As not all amebae respond typically to the pH-changes, the effectiveness of this influence seems to depend on the physiological state of the amebae. Heard & Seaman [1 I] showed modifications in the stability of Exptl CeII Res 80 (1973)
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Batueva, 1 V, Cytology 6 (1964) 209. - Ibid 7 (1965) 188. Batueva, I V & Lev, A A, Cytology 9 (1967) 680. Bingley, M S, Exptl cell res 34 (1964) 266. - J exptl biol 45 (1966) 251. Braatz-Schade, K & Stockem, W, Arch Protistenk 114 (1972) 272. Brandt, P W & Freeman, A R, Science 155 (1967) 582. Brewer. J E & Bell. L G E. J cell sci 4 (1969) . , 17. - Ibid 7 (1970) 549. Haberey, M & Stockem, W, Mikrokosmos 60 (1971) 33. Heard. D H & Seaman, G V F, J gen physiol 43 (1960) 653. Jeon, K W & Bell, L G E, Exptl cell res 27 (1962) 350. Korohoda, W, Acta protozool 11 (1972) 333. Korohoda. W. Ambrose, E J & Forrester, J A, Folia biol i5 (i967) 371. Wolpert, L & Gingell, D, Symp sot exptl biol 22 (1968) 169.
Received February 1, 1973 Revised version received May 7, 1973