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Further investigations into membrane potentials in amoebae
Experimental
Cell Research 43, l-12 (1966)
FURTHER
INVESTIGATIONS INTO MEMBRANE POTENTIALS IN AMOEBAE1 M. S. BINGLEY
RAF
Institute
of Aviation Hampshire,
Medicine, England
Farnborough,
Received December 8, 19fX?
MEMBRANE potentials recorded from the tips of streaming pseudopods in Amoeba proteus were found on average to be some 34 mV less negative than those recorded from the rear region (Bingley and Thompson [5]). These authors also found that potentials recorded in the rear regions of moving amoebae tended to increase in magnitude as the cells streamed forward over the electrode. Cytoplasmic streaming appeared to be orientated towards the positive region of a potential gradient within the cytoplasm of the cell. Cytoplasmic streaming into pseudopodia was often reversed by inadvertent mechanical stimulation, produced by the insertion of a microelectrode into the pseudopod tip. This reversal was always accompanied by an increase of negative potential in the region of the pseudopod [2]. Kamiya [9] and Tasaki and Kamiya [lo] were unable to find this intracellular potential difference in Amoeba proteus when measuring membrane potentials in cells within their double chamber and expressed doubts as to the meaning of membrane potentials in amoebae when these were measured in the presence of electrode tip potentials. Batueva [l ] overcame the problem of electrode tip potential by culturing Amoeba proteus in media in which the normal ion concentration was increased by a factor of ten. Bingley and Thompson’s [5] findings were confirmed as far as the potential difference between the tip of the pseudopod and the middle region is concerned, but a reduction of potential when the electrode was inserted into the uroid was found. An explanation was put forward to account for the lower pseudopod potential based on the possibility that the membrane in this region did not completely seal round the electrode. Thus the full membrane potential would be shunted by leakage paths. This hypothesis was based on the observation 1 This was given in the form of a communication Hill, England, on 5th November 1965. I acknowledge publish Fig. 3. 2 Revised version received April 13, 1966. 1 - 861806
in the Physiological Society Meeting at Mill the permission of the Physiological Society to
Experimental
Cell Research 43
2
M. S. Bingley
that the increase of electrode resistance recorded by a microelectrode which penetrated a pseudopod was less than that when the electrode was in the rear region. Bingley [3] described a technique to enable a saline filled glass microelectrode to be used in Chalkley’s medium, Chalkely [6], without the presence of electrode tip potentials. This was essentially a method of lowering electrode resistance and tip potential. With these electrodes it was possible to record membrane potentials without damage to the cells and to maintain a constant baseline or zero point throughout the experiment when the electrode was withdrawn from the cell. Further analysis of this technique was made. It was found that a microelectrode, treated, by passing the tip through agar gel, would show no tip potential change during an extreme change in the concentration of ions surrounding the tip [4]. Since there is a considerable difference of opinion as to whether there really is a potential difference existing within the cytoplasm of Amoeba proteus between an advancing pseudopod and the rear region, it was decided to repeat some of the experiments described by Bingley and Thompson [5] in which the membrane potential was measured in an advancing pseudopod and also in the rear region of the same cell. These potentials have been recorded in the form of photographic records. At the same time electrode resistance has been monitored throughout the penetrations to see whether there is any substance in the shunting hypothesis of Batueva [l] concerning the potential difference between the advancing pseudopod and the middle or rear region, It is well known that amoebae are sensitive to mechanical stimulation. Considerable changes in membrane potential can be induced in this way. It is possible that the diversity of potential measurements in amoebae may be due to experimental technique involving mechanical constraint or restriction of the cells. Tasaki and Kamiya [lo] used a double chamber which constricted the cell in the middle region. Where potential differences have been found it is noticeable that the cells have been free and unconstricted [l, 2, 51. METHODS Cultures of Amoeba proteus were grown in Chalkley’s medium [6]. They were fed on Tetrahymena pyriformis in mass culture four times a week. The composition of Chalkley’s medium is: 1.37 mM NaCl, 0.027 mM KCl, 0.047 mM NaHCO,, 0.007 mlM Na,HPO,, 0.007 mM CaHPO,. All reagents used were of analar quality. Glass distilled water was used throughout. Experimental
Cell Research 43
Potentials
in amoebae
3
The experimental technique for recording membrane potentials has already been described [3]. Essentially, amoebae were introduced into an experimental chamber, open at the top, containing dilute Chalkley’s solution. The amoebae attached themselves to the bottom surface of this chamber where they streamed freely. Membrane potentials were recorded by means of saline filled glass microelectrodes (3.0 M KCl) which were manipulated with Leitz micromanipulators. These were connected to a high input impedance, low grid current cathode follower system. This device has been cathodally screened [7] and will record a square wave of 1 kc across a 10 Megohm resistor with very little distortion. Potentials were recorded by means of a camera attached to an oscilloscope whose time base was driven by a rotating potentiometer. By this means it was possible to produce linear sweeps as slow as one cycle in five minutes. A second oscilloscope operating a calibrated fast time base enabled repetitive phenomena to be observed accurately which would otherwise not be possible using the slow mechanical time base. Electrode resistance was continuously monitored throughout the experiments by means of pulses fed at 5 set or 1 set intervals through a 20 Megohm high stability k 1 per cent resistance to the microelectrode via an 0.05 PF ceramic capacitor. By the substitution of known values of resistance for the microelectrode it was possible to construct pulse attenuation curves, and to determine from these, electrode resistance. Since the internal conductivity of amoeba cytoplasm is as yet not accurately known the increase of resistance recorded by the electrode after insertion through the membrane cannot be ascribed to any particular component in the system. Electrode resistance recorded when the electrode is out of the cell will be referred to as R.E.
RESULTS The first series of experiments involved penetrating pseudopods with a single microelectrode and endeavouring to cause a reversal of forward streaming by mechanical stimulation with the microelectrode. At the same time membrane potentials were continuously monitored and recorded. Fig. 1 illustrates a microelectrode inserted into a forward streaming pseudopod. A low membrane potential was recorded while the pseudopod continued to stream forward. Gentle vibration of the bench caused the streaming to cease abruptly. An immediate rise in potential can be seen to accompany the cessation of streaming. (NB. This effect contrasts with the fall in potential of the rear region after mechanical stimulation as shown in Fig. 4.) When the electrode was withdrawn there was a return to the baseline as can be seen from the figure. A second amoeba was chosen and this experiment was repeated. Fig. 2 illustrates the membrane potential recorded by a microelectrode inserted into an advancing pseudopod of this second cell. Inadvertent mechanical stimulation produced by microelectrode insertion caused the Experimental
Cell Research 43
M. S. Bingley
O&,” Fig. 1. Fig. l.-Membrane potentials recorded from the tip of an advancing potentials in pseudopod; b, streaming ceased.
Fig. 2. pseudopod.
a, Membrane
Fig. 2.-Membrane potentials recorded from the tip of an advancing pseudopod. a, membrane potentials in pseudopod; b, electrode resistance spikes; c, streaming ceases; d, streaming reversed.
streaming within the pseudopod to cease and then to reverse its direction. In this case the whole cell reversed its direction of locomotion. Coincident with this there was a rise in the negativity recorded within the pseudopod. On withdrawal of the microelectrode there was a return to the baseline with no shift in potential. Electrode resistance spikes indicate electrode resistance at the end of the experiment (R.E. in excess of 10 Megohms). In the next series of experiments membrane potentials were recorded from the tip of an advancing pseudopod and the rear region of the same cell using one microelectrode. At the same time electrode resistance was continuously monitored while the electrode was within and without the cytoplasm. Mechanical stimulation of the membrane was also applied at certain points in some experiments. Fig. 3 illustrates membrane potentials recorded from an advancing pseudopod by an electrode of resistance 2.6 Megohms. While the pseudopod was streaming forward a membrane potential of -18 mV was recorded. There was an increase in the resistance recorded by the electrode of 8.4 Megohms (R.E. =2.6 Megohms -+ll Megohms). When streaming reversed in Experimental
Cell Research 43
Potentials
3
in amoebae
o-
mV -5OL
Fig. 3.
Fig. 4.
Fig. 3.-Membrane potentials recorded from the tip of an advancing pseudopod and the rear region of the same amoeba with one electrode. a, membrane potentials in pseudopod during forward streaming; b, backward streaming begins in pseudopod; c, electrode withdrawn; d, membrane potentials in rear region; e, mechanical stimulation. Fig. 4.-Membrane potentials recorded from the middle region of an advancing pseudopod and the middle region of the same amoeba with one electrode. a, Membrane potentials in pseudopod middle region; b, electrode withdrawn; c, membrane potentials in middle region; d, mechanical stimulation by probe.
the pseudopod there was an immediate jump of potential from -18 mV to - 50 mV and a reduction in the resistance recorded by the electrode system (11 Megohms +5.4 Megohms). On removal of the electrode from the pseudopod the resistance recorded by the microelectrode returned to its former value (R.E. =2.6 Megohms). The microelectrode was now inserted into the rear region in which there was steady forward streaming. A membrane potential of - 50 mV was recorded which steadily increased to - 70 mV. During this second insertion mechanical vibration was applied to the electrode four times. This caused depolarization of up to 30 mV followed by a 5 set recovery period. When the microelectrode was removed there was a return to the original baseline and the electrode resistance returned to 2.6 Megohms. During the period of potential increase while the electrode was within the rear region there was a steady increase of resistance recorded by the microelectrode. Experimental
Cell Research 43
M. S. Bingley
t50 mv. 0
-50.
go t6iat;-l Fig. 5. Fig 5.-Membrane potentials recorded from an advancing pseudopod and the middle region of the same amoeba with one electrode. a, Membrane potentials in rear region; b, electrode withdrawn; c, membrane potentials in pseudopod. Fig. 6.-Membrane potentials recorded from the tip of an advancing pseudopod and the rear region of the same amoeba using one electrode. a, membrane potentials in pseudopod; b, membrane potentials in rear region; c, electrode breaks; d, cell membrane expands; e, electrode withdrawn.
Fig. 4 illustrates two potentials recorded from a single cell. The first was recorded from the middle region of an advancing pseudopod with a microelectrode of resistance 3.0 Megohms. An initial potential of -40 mV was recorded which steadily rose to -50 mV as the pseudopod continued to stream. The electrode recorded a resistance increase of 2.4 Megohms (R.E. = 3.0 Megohms +5.4 Megohms) which remained constant throughout the experiment while the electrode was within the cytoplasm. The microelectrode was withdrawn and re-inserted into the middle region to record a membrane potential of -43 mV which thereafter rose steadily to -60 mV. At this point the membrane was prodded twice by a microprobe in the region of electrode insertion. This produced depolarizations in the order of 10 mV with a tendency to restore the original potential after stimulation. Throughout this experiment when the microelectrode was withdrawn from the cell there was a return of potential to a constant baseline. Fig. 5 illustrates membrane potentials recorded from the rear region of an Experimental
Cell Research 43
Potentials
7
in amoebae
advancing pseudopod with an electrode of resistance 3.0 Megohms. On penetrating the rear region there was an initial step potential of -20 mV accompanied by an increase of resistance recorded by the electrode of 23 Megohms (R.E. =3.0 Megohms -26 Megohms). This was followed by a rapid increase in membrane potential to - 50 mV and a fall in the resistance (thick recorded by the electrode to 16 Megohms. A period of oscillation region of trace) at approximately 100 c/s followed. This was accompanied by a fall in membrane potential. A rapid rise in potential followed the cessation of oscillation. The membrane potential finally reached a value of -55 mV. The electrode was then withdrawn. During this withdrawal a second step potential was then observed but this time accompanied by a lower resistance value (14.6 Megohms). A second penetration was made and a membrane potential of -24 mV was then recorded from the tip of an advancing pseudopod. The total resistance increase was of the order of 11.6 Megohms on insertion. On withdrawal of the microelectrode there was a return to zero potential. Throughout the experiment while the electrode was outside the cell the baseline was constant and electrode resistance was constant at 3.0 Megohms. Observations have been made on the behaviour of amoebae when penetrated by a microelectrode which has broken while inside the cell [2, 31. This breakage has often been accompanied by a reversal of membane potential. At the same time the cell expands to about five times its original size and the membrane appears to “lift off” in the same manner as described by Goldacre [S]. The membrane potential slowly subsides to zero as the membrane disintegrates. Fig. 6 illustrates the sequence of events following the breakage of the tip of a microelectrode inserted into the cytoplasm of an amoeba. The first penetration of a 3.0 Megohm electrode into the tip of an advancing pseudopod was normal and yielded a membrane potential of -23 mV. There was an increase in resistance recorded by the microelectrode to 9.0 Megohms. The second penetration in the rear region produced an initial negative spike of -40 mV. This was followed by a total reduction of membrane potential to zero. Total resistance recorded by the electrode fell to 1.0 Megohm. The cell rapidly increased in size and the membrane appeared to “lift off”. This was accompanied by a reversal of membrane potential to +73 mV. The potential subsided as the membrane slowly disintegrated. Throughout this experiment there was a return to a constant baseline when the microelectrode was out of the cytoplasm. The microelectrode resistance had been reduced to 1.0 Megohm at the end of the experiment. Experimental
Cell Research 43
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M. S. Bingley
Table I gives the means and standard deviations pseudopods and 12 recordings from the rear region. TABLE
of 17 recordings
from
I. Membrane potentials in mV recorded from two regions of Amoeba proteus.
Average
Pseudopod
Rear region
-30 -30 -34 -20 -11 -29 -58 -18 -27 -24 -24 -20 -23 -22 -21 -6 -20
-95 -80
-24.5
-72 -54 -62 -62 -72 -60 -76 -45 -50 -43
S.D.
22.7
- 64.2
S.D.
k4.4
DISCUSSION
In addition to previous evidence [ 1, 51 these examples illustrate the presence of a potential existing between the tip of an advancing pseudopod and the rear region. Low potentials are only recorded from the tips of advancing pseudopods. Inadvertent mechanical stimulation or deep penetration causes the pseudopod to cease streaming and often to retract and reverse streaming direction. In all cases this is accompanied by a rapid, almost “step like”, rise in negativity. Batueva [ 1 ] contends that low potentials in pseudopods may be due to failure of the membrane to seal round the microelectrode so that shunting paths would be set up. In these experiments a pulse was applied to the microelectrode to monitor electrode resistance when out of the cell, and an increase of resistance was found when the cell was entered. A leakage path would be Experimental
Cell Research 43
Potentials
9
in amoebae
shown as a decrement of this increase of resistance when the microelectrode is within the pseudopod compared to its value when in the rear region but the figures show no sign of this. On the contrary the resistance measured by the electrode when in the pseudopod is either equal or sometimes greater than when in the rear region. Therefore it seems unlikely that the low potentials can be explained on this assumption. It must be remembered that Batueva’s [l] experiments were made on cells in an ionic environment ten times more concentrated than that in which these experiments were done so that shunting might well have been increased. Improvements in micromanipulation technique have enabled reliable membrane potentials to be recorded from amoebae with microelectrodes of lower resistance than those used and described by Bingley [3, 41. Furthermore these improvements enabled a microelectrode to be placed in the tip of an advancing pseudopod with the result that an average membrane potential of -24.5 mV (Table I) was recorded and this can be compared with the previous work of Bingley and Thompson [5] in which an average membrane potential of -36 mV was recorded in the pseudopod. In the latter case, it was not possible to insert the microelectrode into the tip of the advancing pseudopod. In other words, the closer the microelectrode is to the tip of the advancing pseudopod, the lower is the recorded potential. Although the membrane is probably responsible for the main component of the increase of electrode resistance when within the cell, this is as yet uncertain. Tasaki and Kamiya [lo] advance the possibility that the intracellular potassium is almost fully bound to cytoplasmic components, and exhibits little osmotic activity or electrical conductivity. Part of their evidence for this relies on the observation that freshwater amoebae are not fully turgid and require little pressure to deform their membranes. A comparison is made between this situation and that in certain freshwater algal cells which exhibit extreme turgor. However they have overlooked the fact that there is a contractile vacuole which is pumping water out of the cell in amoebae, whereas in algae there is not such a system. Ideally two microelectrodes should be inserted into an advancing pseudopod to record membrane resistance in this area, one electrode to pass current and the other voltage; but it is unlikely that the pseudopod would continue to stream forward with two microelectrodes inserted into it. Membrane potentials recorded from the rear region in Fig. 3 and 4 confirm earlier findings of Bingley and Thompson [5] and observations of Batueva [l] that there is a steady rise in potential during forward streaming. Batueva [l ] described similar results but found a change in electrode tip potential Experimental
Cell Research 43
10
M. S. Bingley
withdrawing the electrode. In all these experiments the baseline remained constant throughout the experiment. Also in Fig. 3 it can be seen that there was an increase in the value of total resistance recorded bythe electrode during the potential rise. This again agrees with Batueva’s findings. Shifts in the baseline potential can be induced by pushing a microelectrode right through the cell. The electrode is blocked and there is an increase of electrode resistance which is accompanied by a secondary negative tip potential when the electrode is removed from the cell. Mechanical stimulation in the rear region of the cell produced depolarizations of the order of 30 mV. This is followed by a slow recovery period often of the order of 5 sec. Mechanical stimulation can be either by probe in a region adjacent to that of the electrode insertion, or it can be induced by vibrating the microelectrode itself. It is not easy to induce mechanical stimulation in a pseudopod by probe without displacing the microelectrode so that no voltage is recorded. However, it is possible to vibrate a microelectrode inserted into the tip of an advancing pseudopod and thus to produce mechanical stimulation in this region. Always this is accompanied by a rapid rise in potential. It is possible that these observations are the electrical counterpart of Goldacre’s [S] observations that when a pseudopod is stimulated at the tip there is a re-direction of streaming and that when the rear region of an amoeba is probed a pseudopod is often produced or elicited at that point. Thus in the former there is a hyperpolarization which would result in a reversal of streaming. In the latter there is a depolarization which would initiate a new pseudopod. Step potentials are sometimes encountered while penetrating the rear regions of amoebae. Since it is known that amoebae respond to touch in this region by depolarization it is likely that initial microelectrode penetration leads to membrane depolarization. This would be followed by repolarization as the electrode is inserted further into the cytoplasm. Fig. 6 illustrates an interesting phenomenon frequently observed by Bingley [2, 31, that when a microelectrode breaks during penetration there is a rapid depolarization of membrane potential followed by a positive excursion. This positive excursion is accompanied by a swelling of the cell to about five times its original volume and the appearance of large clear areas in the cytoplasm. In many cases this reaction has been observed to start at the point of electrode insertion and to spread over the whole cell membrane. It has also been noted that if the microelectrode is withdrawn quickly in a few cases the original form of the membrane can reassert itself and the small bleb disappears. It is possible that this is a form of propagated reaction spreading Experimental
Cell Research 43
Potentials
11
in amoebae
across the cell’s membrane akin to that which takes place during the passage of an action potential in the nerve cell. It is tempting to think that the potential shown in Fig. 6 is in reality a very primitive form of action potential, the difference in this case being the inability of the cell membrane to recover and to restore full membrane negative potential as the nerve cell does. However, such considerations are as yet only extremely speculative. The following detailed observations have been made on the morphological results of electrode insertion. (1) The cytoplasm streams move instantly towards the point of electrode insertion immediately the potential recorded by the electrode reverses from negative or neutral to positive. (2) If the process is not allowed to proceed too far and the electrode is removed, new pseudopods are often initiated at the point where the electrode has been inserted. (3) The visible change in the membrane is very similar to that observed when applying a positive potential to an electrode inserted in the rear region of an amoeba for a long period. The membrane changes from an irregular outline to that of a smooth one. This change spreads slowly outward over the whole cell from the point of electrode insertion. It is of interest that regions of amoebae which are either known or calculated to be positive, relative to other regions, do assume certain visible characteristics which are very similar to those of an advancing pseudopod. One of these is a smooth membrane with a clear region (hyaline layer?) separating it from the internal cytoplasm. Preliminary experiments involving the penetration of a cell with two microelectrodes, one applying a current pulse and the other measuring voltage, have yielded tentative figures of 2.0 Megohms for possible membrane resistance. However, insertion of the current electrode depolarized the cell and we must regard this figure as being very preliminary and referring to a cell in a depolarized state. The evidence points to the possibility of there being a potential gradient within freely streaming amoebae. The precise nature of the relationship of cytoplasmic streaming to this potential gradient is as yet unknown. SUMMARY
1. The present work confirms the findings of Bingley and Thompson and Batueva that membrane potentials recorded from the tip of an advancing pseudopod are less negative than those recorded from the rear region. Photographic records are presented. Experimental
Cell Research 43
M. S. Bingley
12
2. Pseudopods streaming forward exhibit low potentials -24.5 mV S.D. i 2.7 compared to the rear region -64.2 mV S.D. k4.4. 3. Mechanical stimulation in the rear region produced a partial depolarization of the membrane. 4. Breakage of a microelectrode while inside the cell was often followed by a positive membrane potential excursion of up to +70 mV. The cell expanded and water appeared to enter the cell. Photographic traces show the course of this potential change which shows some of the characteristics of an action potential. 5. Electrode resistance was continuously monitored while membrane potentials mere recorded. Increases in this during penetration suggest that there is only one resistive barrier, probably the cell membrane through which the electrode passes, whether it is in the pseudopod or rear region. This work was done under the auspices of a Government Senior Research Fellowship at the RAF Institute of Aviation Medicine. I am grateful to Air Commodore W. K. Stewart, B.Sc., M.B., Ch.B., for help and encouragement. Likewise I would like to acknowledge Professor H. Holter of the Carlsberg Laboratorium, Copenhagen; Dr J. W. D. Beament, F.R.S., Department of Zoology, University of Cambridge, and Dr D. A. T. Dick, Department of Human Anatomy, University of Oxford. My thanks also to Mr D. R. Baillie of the Photographic Section, Mr C. W. Baker of the Workshops, and Mr S. Hunter, of the RAF Institute of Aviation Medicine. Lastly I would like to acknowledge the help of Mr Veal1 of Ernst Leitz Ltd. of London, for the help he gave in the initial setting up of the optical and micromanipulative equipment. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
BATUEVA, I. V., Tsitologyia, 6, 209-212 (1964). BINGLEY, M. S., PhD Thesis, London University 1962. -Exptl Cell Res. 34, 266 (1964). -Nature 202, 1218 (1964). BINGLEY, M. S. and Thompson, C. M., J. Tkeoret. Biol. 2, 16 (1962). CHALKLEY, H. W., Science 71, 442 (1930). DONALDSON, P. E. K., Electronic apparatus for biological research. Butterworth, 1958. GOLDACRE, Fi. J., Symp. Exptl Biol. 6, 128 (1952). KAMIYA, N., Motive force of endoplasmic streaming in amoebae. Primitive Motile Systems in Cell Biology, p. 257. Academic Press, New York, 1964. 10. TASAKI, I. and KAMIYA, N., J. Cell. Comp. Pkysiol. 63, 3 (1964).
Experimental
Cell Research 43
Cell Research 43, l-12 (1966)
FURTHER
INVESTIGATIONS INTO MEMBRANE POTENTIALS IN AMOEBAE1 M. S. BINGLEY
RAF
Institute
of Aviation Hampshire,
Medicine, England
Farnborough,
Received December 8, 19fX?
MEMBRANE potentials recorded from the tips of streaming pseudopods in Amoeba proteus were found on average to be some 34 mV less negative than those recorded from the rear region (Bingley and Thompson [5]). These authors also found that potentials recorded in the rear regions of moving amoebae tended to increase in magnitude as the cells streamed forward over the electrode. Cytoplasmic streaming appeared to be orientated towards the positive region of a potential gradient within the cytoplasm of the cell. Cytoplasmic streaming into pseudopodia was often reversed by inadvertent mechanical stimulation, produced by the insertion of a microelectrode into the pseudopod tip. This reversal was always accompanied by an increase of negative potential in the region of the pseudopod [2]. Kamiya [9] and Tasaki and Kamiya [lo] were unable to find this intracellular potential difference in Amoeba proteus when measuring membrane potentials in cells within their double chamber and expressed doubts as to the meaning of membrane potentials in amoebae when these were measured in the presence of electrode tip potentials. Batueva [l ] overcame the problem of electrode tip potential by culturing Amoeba proteus in media in which the normal ion concentration was increased by a factor of ten. Bingley and Thompson’s [5] findings were confirmed as far as the potential difference between the tip of the pseudopod and the middle region is concerned, but a reduction of potential when the electrode was inserted into the uroid was found. An explanation was put forward to account for the lower pseudopod potential based on the possibility that the membrane in this region did not completely seal round the electrode. Thus the full membrane potential would be shunted by leakage paths. This hypothesis was based on the observation 1 This was given in the form of a communication Hill, England, on 5th November 1965. I acknowledge publish Fig. 3. 2 Revised version received April 13, 1966. 1 - 861806
in the Physiological Society Meeting at Mill the permission of the Physiological Society to
Experimental
Cell Research 43
2
M. S. Bingley
that the increase of electrode resistance recorded by a microelectrode which penetrated a pseudopod was less than that when the electrode was in the rear region. Bingley [3] described a technique to enable a saline filled glass microelectrode to be used in Chalkley’s medium, Chalkely [6], without the presence of electrode tip potentials. This was essentially a method of lowering electrode resistance and tip potential. With these electrodes it was possible to record membrane potentials without damage to the cells and to maintain a constant baseline or zero point throughout the experiment when the electrode was withdrawn from the cell. Further analysis of this technique was made. It was found that a microelectrode, treated, by passing the tip through agar gel, would show no tip potential change during an extreme change in the concentration of ions surrounding the tip [4]. Since there is a considerable difference of opinion as to whether there really is a potential difference existing within the cytoplasm of Amoeba proteus between an advancing pseudopod and the rear region, it was decided to repeat some of the experiments described by Bingley and Thompson [5] in which the membrane potential was measured in an advancing pseudopod and also in the rear region of the same cell. These potentials have been recorded in the form of photographic records. At the same time electrode resistance has been monitored throughout the penetrations to see whether there is any substance in the shunting hypothesis of Batueva [l] concerning the potential difference between the advancing pseudopod and the middle or rear region, It is well known that amoebae are sensitive to mechanical stimulation. Considerable changes in membrane potential can be induced in this way. It is possible that the diversity of potential measurements in amoebae may be due to experimental technique involving mechanical constraint or restriction of the cells. Tasaki and Kamiya [lo] used a double chamber which constricted the cell in the middle region. Where potential differences have been found it is noticeable that the cells have been free and unconstricted [l, 2, 51. METHODS Cultures of Amoeba proteus were grown in Chalkley’s medium [6]. They were fed on Tetrahymena pyriformis in mass culture four times a week. The composition of Chalkley’s medium is: 1.37 mM NaCl, 0.027 mM KCl, 0.047 mM NaHCO,, 0.007 mlM Na,HPO,, 0.007 mM CaHPO,. All reagents used were of analar quality. Glass distilled water was used throughout. Experimental
Cell Research 43
Potentials
in amoebae
3
The experimental technique for recording membrane potentials has already been described [3]. Essentially, amoebae were introduced into an experimental chamber, open at the top, containing dilute Chalkley’s solution. The amoebae attached themselves to the bottom surface of this chamber where they streamed freely. Membrane potentials were recorded by means of saline filled glass microelectrodes (3.0 M KCl) which were manipulated with Leitz micromanipulators. These were connected to a high input impedance, low grid current cathode follower system. This device has been cathodally screened [7] and will record a square wave of 1 kc across a 10 Megohm resistor with very little distortion. Potentials were recorded by means of a camera attached to an oscilloscope whose time base was driven by a rotating potentiometer. By this means it was possible to produce linear sweeps as slow as one cycle in five minutes. A second oscilloscope operating a calibrated fast time base enabled repetitive phenomena to be observed accurately which would otherwise not be possible using the slow mechanical time base. Electrode resistance was continuously monitored throughout the experiments by means of pulses fed at 5 set or 1 set intervals through a 20 Megohm high stability k 1 per cent resistance to the microelectrode via an 0.05 PF ceramic capacitor. By the substitution of known values of resistance for the microelectrode it was possible to construct pulse attenuation curves, and to determine from these, electrode resistance. Since the internal conductivity of amoeba cytoplasm is as yet not accurately known the increase of resistance recorded by the electrode after insertion through the membrane cannot be ascribed to any particular component in the system. Electrode resistance recorded when the electrode is out of the cell will be referred to as R.E.
RESULTS The first series of experiments involved penetrating pseudopods with a single microelectrode and endeavouring to cause a reversal of forward streaming by mechanical stimulation with the microelectrode. At the same time membrane potentials were continuously monitored and recorded. Fig. 1 illustrates a microelectrode inserted into a forward streaming pseudopod. A low membrane potential was recorded while the pseudopod continued to stream forward. Gentle vibration of the bench caused the streaming to cease abruptly. An immediate rise in potential can be seen to accompany the cessation of streaming. (NB. This effect contrasts with the fall in potential of the rear region after mechanical stimulation as shown in Fig. 4.) When the electrode was withdrawn there was a return to the baseline as can be seen from the figure. A second amoeba was chosen and this experiment was repeated. Fig. 2 illustrates the membrane potential recorded by a microelectrode inserted into an advancing pseudopod of this second cell. Inadvertent mechanical stimulation produced by microelectrode insertion caused the Experimental
Cell Research 43
M. S. Bingley
O&,” Fig. 1. Fig. l.-Membrane potentials recorded from the tip of an advancing potentials in pseudopod; b, streaming ceased.
Fig. 2. pseudopod.
a, Membrane
Fig. 2.-Membrane potentials recorded from the tip of an advancing pseudopod. a, membrane potentials in pseudopod; b, electrode resistance spikes; c, streaming ceases; d, streaming reversed.
streaming within the pseudopod to cease and then to reverse its direction. In this case the whole cell reversed its direction of locomotion. Coincident with this there was a rise in the negativity recorded within the pseudopod. On withdrawal of the microelectrode there was a return to the baseline with no shift in potential. Electrode resistance spikes indicate electrode resistance at the end of the experiment (R.E. in excess of 10 Megohms). In the next series of experiments membrane potentials were recorded from the tip of an advancing pseudopod and the rear region of the same cell using one microelectrode. At the same time electrode resistance was continuously monitored while the electrode was within and without the cytoplasm. Mechanical stimulation of the membrane was also applied at certain points in some experiments. Fig. 3 illustrates membrane potentials recorded from an advancing pseudopod by an electrode of resistance 2.6 Megohms. While the pseudopod was streaming forward a membrane potential of -18 mV was recorded. There was an increase in the resistance recorded by the electrode of 8.4 Megohms (R.E. =2.6 Megohms -+ll Megohms). When streaming reversed in Experimental
Cell Research 43
Potentials
3
in amoebae
o-
mV -5OL
Fig. 3.
Fig. 4.
Fig. 3.-Membrane potentials recorded from the tip of an advancing pseudopod and the rear region of the same amoeba with one electrode. a, membrane potentials in pseudopod during forward streaming; b, backward streaming begins in pseudopod; c, electrode withdrawn; d, membrane potentials in rear region; e, mechanical stimulation. Fig. 4.-Membrane potentials recorded from the middle region of an advancing pseudopod and the middle region of the same amoeba with one electrode. a, Membrane potentials in pseudopod middle region; b, electrode withdrawn; c, membrane potentials in middle region; d, mechanical stimulation by probe.
the pseudopod there was an immediate jump of potential from -18 mV to - 50 mV and a reduction in the resistance recorded by the electrode system (11 Megohms +5.4 Megohms). On removal of the electrode from the pseudopod the resistance recorded by the microelectrode returned to its former value (R.E. =2.6 Megohms). The microelectrode was now inserted into the rear region in which there was steady forward streaming. A membrane potential of - 50 mV was recorded which steadily increased to - 70 mV. During this second insertion mechanical vibration was applied to the electrode four times. This caused depolarization of up to 30 mV followed by a 5 set recovery period. When the microelectrode was removed there was a return to the original baseline and the electrode resistance returned to 2.6 Megohms. During the period of potential increase while the electrode was within the rear region there was a steady increase of resistance recorded by the microelectrode. Experimental
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M. S. Bingley
t50 mv. 0
-50.
go t6iat;-l Fig. 5. Fig 5.-Membrane potentials recorded from an advancing pseudopod and the middle region of the same amoeba with one electrode. a, Membrane potentials in rear region; b, electrode withdrawn; c, membrane potentials in pseudopod. Fig. 6.-Membrane potentials recorded from the tip of an advancing pseudopod and the rear region of the same amoeba using one electrode. a, membrane potentials in pseudopod; b, membrane potentials in rear region; c, electrode breaks; d, cell membrane expands; e, electrode withdrawn.
Fig. 4 illustrates two potentials recorded from a single cell. The first was recorded from the middle region of an advancing pseudopod with a microelectrode of resistance 3.0 Megohms. An initial potential of -40 mV was recorded which steadily rose to -50 mV as the pseudopod continued to stream. The electrode recorded a resistance increase of 2.4 Megohms (R.E. = 3.0 Megohms +5.4 Megohms) which remained constant throughout the experiment while the electrode was within the cytoplasm. The microelectrode was withdrawn and re-inserted into the middle region to record a membrane potential of -43 mV which thereafter rose steadily to -60 mV. At this point the membrane was prodded twice by a microprobe in the region of electrode insertion. This produced depolarizations in the order of 10 mV with a tendency to restore the original potential after stimulation. Throughout this experiment when the microelectrode was withdrawn from the cell there was a return of potential to a constant baseline. Fig. 5 illustrates membrane potentials recorded from the rear region of an Experimental
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Potentials
7
in amoebae
advancing pseudopod with an electrode of resistance 3.0 Megohms. On penetrating the rear region there was an initial step potential of -20 mV accompanied by an increase of resistance recorded by the electrode of 23 Megohms (R.E. =3.0 Megohms -26 Megohms). This was followed by a rapid increase in membrane potential to - 50 mV and a fall in the resistance (thick recorded by the electrode to 16 Megohms. A period of oscillation region of trace) at approximately 100 c/s followed. This was accompanied by a fall in membrane potential. A rapid rise in potential followed the cessation of oscillation. The membrane potential finally reached a value of -55 mV. The electrode was then withdrawn. During this withdrawal a second step potential was then observed but this time accompanied by a lower resistance value (14.6 Megohms). A second penetration was made and a membrane potential of -24 mV was then recorded from the tip of an advancing pseudopod. The total resistance increase was of the order of 11.6 Megohms on insertion. On withdrawal of the microelectrode there was a return to zero potential. Throughout the experiment while the electrode was outside the cell the baseline was constant and electrode resistance was constant at 3.0 Megohms. Observations have been made on the behaviour of amoebae when penetrated by a microelectrode which has broken while inside the cell [2, 31. This breakage has often been accompanied by a reversal of membane potential. At the same time the cell expands to about five times its original size and the membrane appears to “lift off” in the same manner as described by Goldacre [S]. The membrane potential slowly subsides to zero as the membrane disintegrates. Fig. 6 illustrates the sequence of events following the breakage of the tip of a microelectrode inserted into the cytoplasm of an amoeba. The first penetration of a 3.0 Megohm electrode into the tip of an advancing pseudopod was normal and yielded a membrane potential of -23 mV. There was an increase in resistance recorded by the microelectrode to 9.0 Megohms. The second penetration in the rear region produced an initial negative spike of -40 mV. This was followed by a total reduction of membrane potential to zero. Total resistance recorded by the electrode fell to 1.0 Megohm. The cell rapidly increased in size and the membrane appeared to “lift off”. This was accompanied by a reversal of membrane potential to +73 mV. The potential subsided as the membrane slowly disintegrated. Throughout this experiment there was a return to a constant baseline when the microelectrode was out of the cytoplasm. The microelectrode resistance had been reduced to 1.0 Megohm at the end of the experiment. Experimental
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M. S. Bingley
Table I gives the means and standard deviations pseudopods and 12 recordings from the rear region. TABLE
of 17 recordings
from
I. Membrane potentials in mV recorded from two regions of Amoeba proteus.
Average
Pseudopod
Rear region
-30 -30 -34 -20 -11 -29 -58 -18 -27 -24 -24 -20 -23 -22 -21 -6 -20
-95 -80
-24.5
-72 -54 -62 -62 -72 -60 -76 -45 -50 -43
S.D.
22.7
- 64.2
S.D.
k4.4
DISCUSSION
In addition to previous evidence [ 1, 51 these examples illustrate the presence of a potential existing between the tip of an advancing pseudopod and the rear region. Low potentials are only recorded from the tips of advancing pseudopods. Inadvertent mechanical stimulation or deep penetration causes the pseudopod to cease streaming and often to retract and reverse streaming direction. In all cases this is accompanied by a rapid, almost “step like”, rise in negativity. Batueva [ 1 ] contends that low potentials in pseudopods may be due to failure of the membrane to seal round the microelectrode so that shunting paths would be set up. In these experiments a pulse was applied to the microelectrode to monitor electrode resistance when out of the cell, and an increase of resistance was found when the cell was entered. A leakage path would be Experimental
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Potentials
9
in amoebae
shown as a decrement of this increase of resistance when the microelectrode is within the pseudopod compared to its value when in the rear region but the figures show no sign of this. On the contrary the resistance measured by the electrode when in the pseudopod is either equal or sometimes greater than when in the rear region. Therefore it seems unlikely that the low potentials can be explained on this assumption. It must be remembered that Batueva’s [l] experiments were made on cells in an ionic environment ten times more concentrated than that in which these experiments were done so that shunting might well have been increased. Improvements in micromanipulation technique have enabled reliable membrane potentials to be recorded from amoebae with microelectrodes of lower resistance than those used and described by Bingley [3, 41. Furthermore these improvements enabled a microelectrode to be placed in the tip of an advancing pseudopod with the result that an average membrane potential of -24.5 mV (Table I) was recorded and this can be compared with the previous work of Bingley and Thompson [5] in which an average membrane potential of -36 mV was recorded in the pseudopod. In the latter case, it was not possible to insert the microelectrode into the tip of the advancing pseudopod. In other words, the closer the microelectrode is to the tip of the advancing pseudopod, the lower is the recorded potential. Although the membrane is probably responsible for the main component of the increase of electrode resistance when within the cell, this is as yet uncertain. Tasaki and Kamiya [lo] advance the possibility that the intracellular potassium is almost fully bound to cytoplasmic components, and exhibits little osmotic activity or electrical conductivity. Part of their evidence for this relies on the observation that freshwater amoebae are not fully turgid and require little pressure to deform their membranes. A comparison is made between this situation and that in certain freshwater algal cells which exhibit extreme turgor. However they have overlooked the fact that there is a contractile vacuole which is pumping water out of the cell in amoebae, whereas in algae there is not such a system. Ideally two microelectrodes should be inserted into an advancing pseudopod to record membrane resistance in this area, one electrode to pass current and the other voltage; but it is unlikely that the pseudopod would continue to stream forward with two microelectrodes inserted into it. Membrane potentials recorded from the rear region in Fig. 3 and 4 confirm earlier findings of Bingley and Thompson [5] and observations of Batueva [l] that there is a steady rise in potential during forward streaming. Batueva [l ] described similar results but found a change in electrode tip potential Experimental
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M. S. Bingley
withdrawing the electrode. In all these experiments the baseline remained constant throughout the experiment. Also in Fig. 3 it can be seen that there was an increase in the value of total resistance recorded bythe electrode during the potential rise. This again agrees with Batueva’s findings. Shifts in the baseline potential can be induced by pushing a microelectrode right through the cell. The electrode is blocked and there is an increase of electrode resistance which is accompanied by a secondary negative tip potential when the electrode is removed from the cell. Mechanical stimulation in the rear region of the cell produced depolarizations of the order of 30 mV. This is followed by a slow recovery period often of the order of 5 sec. Mechanical stimulation can be either by probe in a region adjacent to that of the electrode insertion, or it can be induced by vibrating the microelectrode itself. It is not easy to induce mechanical stimulation in a pseudopod by probe without displacing the microelectrode so that no voltage is recorded. However, it is possible to vibrate a microelectrode inserted into the tip of an advancing pseudopod and thus to produce mechanical stimulation in this region. Always this is accompanied by a rapid rise in potential. It is possible that these observations are the electrical counterpart of Goldacre’s [S] observations that when a pseudopod is stimulated at the tip there is a re-direction of streaming and that when the rear region of an amoeba is probed a pseudopod is often produced or elicited at that point. Thus in the former there is a hyperpolarization which would result in a reversal of streaming. In the latter there is a depolarization which would initiate a new pseudopod. Step potentials are sometimes encountered while penetrating the rear regions of amoebae. Since it is known that amoebae respond to touch in this region by depolarization it is likely that initial microelectrode penetration leads to membrane depolarization. This would be followed by repolarization as the electrode is inserted further into the cytoplasm. Fig. 6 illustrates an interesting phenomenon frequently observed by Bingley [2, 31, that when a microelectrode breaks during penetration there is a rapid depolarization of membrane potential followed by a positive excursion. This positive excursion is accompanied by a swelling of the cell to about five times its original volume and the appearance of large clear areas in the cytoplasm. In many cases this reaction has been observed to start at the point of electrode insertion and to spread over the whole cell membrane. It has also been noted that if the microelectrode is withdrawn quickly in a few cases the original form of the membrane can reassert itself and the small bleb disappears. It is possible that this is a form of propagated reaction spreading Experimental
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Potentials
11
in amoebae
across the cell’s membrane akin to that which takes place during the passage of an action potential in the nerve cell. It is tempting to think that the potential shown in Fig. 6 is in reality a very primitive form of action potential, the difference in this case being the inability of the cell membrane to recover and to restore full membrane negative potential as the nerve cell does. However, such considerations are as yet only extremely speculative. The following detailed observations have been made on the morphological results of electrode insertion. (1) The cytoplasm streams move instantly towards the point of electrode insertion immediately the potential recorded by the electrode reverses from negative or neutral to positive. (2) If the process is not allowed to proceed too far and the electrode is removed, new pseudopods are often initiated at the point where the electrode has been inserted. (3) The visible change in the membrane is very similar to that observed when applying a positive potential to an electrode inserted in the rear region of an amoeba for a long period. The membrane changes from an irregular outline to that of a smooth one. This change spreads slowly outward over the whole cell from the point of electrode insertion. It is of interest that regions of amoebae which are either known or calculated to be positive, relative to other regions, do assume certain visible characteristics which are very similar to those of an advancing pseudopod. One of these is a smooth membrane with a clear region (hyaline layer?) separating it from the internal cytoplasm. Preliminary experiments involving the penetration of a cell with two microelectrodes, one applying a current pulse and the other measuring voltage, have yielded tentative figures of 2.0 Megohms for possible membrane resistance. However, insertion of the current electrode depolarized the cell and we must regard this figure as being very preliminary and referring to a cell in a depolarized state. The evidence points to the possibility of there being a potential gradient within freely streaming amoebae. The precise nature of the relationship of cytoplasmic streaming to this potential gradient is as yet unknown. SUMMARY
1. The present work confirms the findings of Bingley and Thompson and Batueva that membrane potentials recorded from the tip of an advancing pseudopod are less negative than those recorded from the rear region. Photographic records are presented. Experimental
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M. S. Bingley
12
2. Pseudopods streaming forward exhibit low potentials -24.5 mV S.D. i 2.7 compared to the rear region -64.2 mV S.D. k4.4. 3. Mechanical stimulation in the rear region produced a partial depolarization of the membrane. 4. Breakage of a microelectrode while inside the cell was often followed by a positive membrane potential excursion of up to +70 mV. The cell expanded and water appeared to enter the cell. Photographic traces show the course of this potential change which shows some of the characteristics of an action potential. 5. Electrode resistance was continuously monitored while membrane potentials mere recorded. Increases in this during penetration suggest that there is only one resistive barrier, probably the cell membrane through which the electrode passes, whether it is in the pseudopod or rear region. This work was done under the auspices of a Government Senior Research Fellowship at the RAF Institute of Aviation Medicine. I am grateful to Air Commodore W. K. Stewart, B.Sc., M.B., Ch.B., for help and encouragement. Likewise I would like to acknowledge Professor H. Holter of the Carlsberg Laboratorium, Copenhagen; Dr J. W. D. Beament, F.R.S., Department of Zoology, University of Cambridge, and Dr D. A. T. Dick, Department of Human Anatomy, University of Oxford. My thanks also to Mr D. R. Baillie of the Photographic Section, Mr C. W. Baker of the Workshops, and Mr S. Hunter, of the RAF Institute of Aviation Medicine. Lastly I would like to acknowledge the help of Mr Veal1 of Ernst Leitz Ltd. of London, for the help he gave in the initial setting up of the optical and micromanipulative equipment. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
BATUEVA, I. V., Tsitologyia, 6, 209-212 (1964). BINGLEY, M. S., PhD Thesis, London University 1962. -Exptl Cell Res. 34, 266 (1964). -Nature 202, 1218 (1964). BINGLEY, M. S. and Thompson, C. M., J. Tkeoret. Biol. 2, 16 (1962). CHALKLEY, H. W., Science 71, 442 (1930). DONALDSON, P. E. K., Electronic apparatus for biological research. Butterworth, 1958. GOLDACRE, Fi. J., Symp. Exptl Biol. 6, 128 (1952). KAMIYA, N., Motive force of endoplasmic streaming in amoebae. Primitive Motile Systems in Cell Biology, p. 257. Academic Press, New York, 1964. 10. TASAKI, I. and KAMIYA, N., J. Cell. Comp. Pkysiol. 63, 3 (1964).
Experimental
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