Membrane Potentials in Amoeba Proteus

The possibility that the initiation of pseudopods together with the direction of cytoplasmic streaming may be induced by local depolarization of the membrane has been advanced from time to time for many years past. But is it difficult to find a direct statement to this effect in the literature. Amici (1818) suggested an electrical theory to account for streaming in plant cells and more recently Kitching (1961) considers the possibility of depolarization of the cell surface initiating contraction. When he discussed Hahnert’s work (Hahnert, 1932) on the response of amoebae to electricity he pointed out that movement of the pseudopods towards the cathode may be an enhancement of ‘local currents associated with local excitation’. It is one thing to put forward a hypothesis but another to obtain convincing measurements which are free from the suspicion of doubt and artifact. Membrane potentials were first seriously measured in Amoeba proteus by Telkes in 1931. Using large electrodes, she obtained low values of membrane potential but produced extremely valuable information on various depolarizing agents such as potassium and sodium chloride. Buchtal & Peterfi (1936) obtained low values of potential for A. proteus. Wolfson (1943) produced convincing values for membrane potential in Chaos chaos and, using electrodes of large diameter, he obtained potentials as high as workers using more modem micro-electrodes. More recently Riddle (1962) working on PeUomyxa carolinensis recorded values of — 90 mV. for the membrane potential and carried out convincing studies on the effect of increasing concentrations of external electrolytes on the membrane potential. Observations were made on the lack of effect of certain metabolic inhibitors on membrane potential. However, all these workers appear to have regarded the cell as being uniform in its electrical properties over its whole surface and to have disregarded the possibility that in a freely streaming amoeba, complete with actively advancing pseudopods showing well-defined hyaline caps, the membrane properties may vary topographically and that this might be reflected in differences of membrane potentials. It must be realized that the advent of micromanipulators capable of inserting a microelectrode into the tip of a fast-moving pseudopod is only relatively recent. Bingley & Thompson (1962), using such a manipulator, were able to test directly certain hypotheses on the possible connexion between cytoplasmic streaming, pseudopod formation and electrical potential changes. They carried out a series of experiments involving direct potential measurement with saline-filled glass microelectrodes and found that the potential in the pseudopod was some 30 mV. less than the rear region, which on average was of the order of — 65 mV. A great deal of subsidiary evidence pointed to the conclusion that cytoplasmic streaming was related to these potential differences. It is of note that these cells were freely streaming and unconstrained in any way. Japanese work, notably that of Kishimoto (1957), indicated a possible relationship between streaming and changes of membrane potential in the slime moulds. Kamiya (1964) and Tasaki & Kamiya (1964) later attempted to confirm the potential difference reported by Bingley & Thompson (1962) and failed to do so, Tasaki being very much concerned with the presence of a circulating current postulated by Bingley & Thompson. Batueva (1965), working on freely moving A. proteus, was able to confirm the presence of a low membrane potential in the pseudopod but explained these observations in terms of short-circuiting of the membrane potential through improper sealing of the membrane round the microelectrode in the region of the pseudopod.

Two problems face modern workers investigating membrane potentials in freshwater A. proteus. The first is the simple fact that these cells live in fresh water and not in saline media, and the second arises from the almost universal use of hyperfine microelectrodes. Incidentally botanists face the same problems, which are complicated by the toughness of plant cell walls. Recently Bingley (19646, 1965 a) investigated the behaviour of hyperfine glass microelectrodes in solutions ranging from dilute Chalklev’s solution on the one hand, which is one-hundredth the molarity of Ringer, to 3·0 M potassium chloride on the other. He came to the conclusion that electrode characteristics such as tip potential and electrode resistance tend to be exaggerated in very dilute solutions and are a function of external ion concentration. Changes in these could well mask bio-electric potentials encountered on passing through the membrane of fresh-water A. proteus. However, the author has never used hyperfine microelectrodes for recording membrane potential in fresh-water amoebae and has always treated his electrodes to reduce resistance and tip potential by widening their tips before insertion (Bingley, 1964a, b, 1965 a). It is also noticeable that earlier workers, notably Telkes (1931), Wolfson (1943) and Riddle (1962), obtained consistent potentials using saline-filled microelectrodes of large diameter (100 μ) on large cells. Bingley (19656) repeated the experiments of Bingley & Thompson (1962) and presented the results in the form of photographic records, laying a great stress on the absence of tip potential and constant base-line. However, all this evidence is not as yet absolutely convincing since we have as yet no concrete proof that when low potentials are encountered in the pseudopod the microelectrode has in reality penetrated the membrane and is not simply indenting the membrane as in the experiments on sea-urchin eggs (Hiramoto, 1959).

This paper is mainly concerned with experiments designed to test the validity of the hypothesis that the low potential recorded from an advancing pseudopod is a genuine membrane potential. In the process other observations have been made and recorded which are relevant to the process of amoeboid locomotion.

Particular attention has been paid to membrane potentials recorded in the rear region of streaming amoebae and to observations made on changes of electrode response while recording these potentials. Previous work on the behaviour of electrodes not inserted into the cell (Bingley, 1964b, 1965 a) has enabled possible interpretations of the state of ions within A. proteus to be put forward.

Cultures of A. proteus were grown in Chalkley’s medium. 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 HIM KC1, 0·047 NaHCO₃, 0·007 mM Na₂HPO₄ 0·007 mM CaHPO₄. All reagents used were of analytical quality. Glass-distilled water was used throughout.

The experimental technique for recording membrane potentials has already been described (Bingley, 1964a).

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-KCI) 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 (Donaldson, 1958) and will record a square wave of 1 kc across a 10 MΩ 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 1 cycle in 5 min. Electrode resistance was continuously monitored throughout the experiments, involving single microelectrode recording by means of pulses fed at 5/sec, or 1 sec. intervals through a known value of resistance to the microelectrode via a capacitor.

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.

Two microelectrodes were inserted into the cell at the same time at opposite ends for the voltage/current experiments. One recorded voltage and the other served to pass current through the membrane in the form of an alternating pulse. This alternating pulse has a fast rise time and a slow exponential decay of 50 milliseconds. Since amoeba membrane has a capacitative element (Tasaki and Lamiya, 1964) measurements made with this technique will be referred to as impedances.

Figure 1  illustrates the experimental situation. An electrode connected directly to a low-resistance source of alternating voltage (see inset waveform) is inserted into the rear region of an amoebae attached to the bottom surface of the experimental chamber. This electrode will pass current through the internal cytoplasm and surface membrane. A 1 MΩ resistance is inserted into the earth connexion to enable the intensity of the current to be measured on an oscilloscope through a cathode follower.

An amoeba can be regarded as a more or less spherical object containing a medium which has a relatively high electrical conductivity bounded by a membrane of low conductivity sitting in a medium of very low electrical conductivity. If a voltagerecording microelectrode picks up a pulsating voltage from a previously implanted current electrode when recording low potentials in the pseudopod, there are grounds for saying that it has passed through a discrete impedance barrier. Only one structure in A. proteus has this property and that is the plasma membrane.

This series of voltage/current experiments was designed to test the hypothesis that the low potential in the tip of a streaming pseudopod is in reality a true membrane potential.

Figure 2  illustrates membrane potentials recorded by the voltage microelectrode inserted into the tip of an advancing pseudopod three times to record a d.c. potential of — 20 mV. The lower trace monitors the current passing through the system via the current electrode both when it is inside the cell and when it is outside. The currentcarrying electrode was the first electrode to be inserted into the cell, in the rear region. The current trace shows an increase of current flowing through the system at this point; likewise there is an increase of alternating voltage appearing on the record produced by the voltage electrode. On penetrating the pseudopod with the voltage electrode a potential of — 20 mV. was recorded ; superimposed on this is the alternating voltage, due to the current electrode, which now shows a 50 % increase in intensity but returns to its original value when the electrode is removed from the pseudopod. During the third penetration of the pseudopod the current electrode was withdrawn from the rear region. The current trace shows a return to its previous level before the current electrode is inserted into the cell. The voltage trace shows a sharp reduction in the alternating voltage recorded at this point. Calculations of membrane impedance yield values of 1 · 4 MΩ.

Figure 3  illustrates potentials recorded from two pseudopods on the same amoeba respectively labelled A and B in the drawing. Throughout the duration of this experiment the current electrode was inserted into the rear region. Again there was an increase of alternating voltage superimposed on the voltage recorded by the voltage electrode when within the pseudopod. The current trace shows a decrease in current flowing through the membrane during penetration of the pseudopod. The drawing below illustrates the relative disposition of pseudopods and micro-electrodes. Membrane impedance as calculated during the penetration of pseudopod A is 9·0 Mil and for pseudopod B, 10·1 MΩ.

The experiment was reversed so that potentials were recorded from the rear region by the voltage electrode ; then the current electrode was inserted into an advancing pseudopod. This would test the assumption that high potentials recorded from the rear are in reality membrane potentials.

Figure 4  illustrates a potential of —65 mV. recorded from the rear region with the voltage electrode. During this recording the current electrode was inserted twice into an advancing pseudopod. The alternating voltage picked up by the voltage electrode increased by over 50 % ; at the same time the cell potential decreased to zero in the first instance and to a few millivolts in the second instance. There was a steady decline in the d.c. component recorded from the rear region during this experiment. Inspection of the current trace during the period when the current electrode was inserted into the pseudopod shows an increase of current flow followed by a return to the original value after the electrode was removed. Of interest is a step potential encountered while the voltage electrode was slowly inserted into the rear region. This was followed by a spike potential whose amplitude was in excess of too mV. ; there was then another rapid rise of potential to a steady level. Membrane impedances associated with the penetration of the current-recording electrode are : for the first penetration, 2·0 MΩ → 1·5 MΩ; and for the second 1·0 MΩ→0·8 MΩ.

Figure 5  illustrates two recordings in the rear region with the voltage electrode recording potentials of — 60 and — 70 mV. respectively. In the first recording the current electrode was inserted into the pseudopod twice, and in the second, once. During these insertions there was a depolarization of the order of 50% of the total d.c. voltage recorded by the voltage electrode, whereas the a.c. component increased by more than 100%. The current trace showed a slight decrease in current flowing through the system during penetration. Calculations of membrane impedance during penetrations yield a constant value of 3·0 MΩ. Again during the initial insertion there was exhibited the characteristic pattern of the step potential followed by a spike exceeding 100 mV. in amplitude.

The one event that is interesting above all others in these last two experiments is a depolarization produced in the rear region by the insertion of the current microelectrode, but not vice versa. It was thought worth while to examine records in which the current microelectrode did not penetrate the membrane either in the pseudopodial or the rear region to see whether this could be a response to touch.

Figure 6  illustrates a potential of-60 mV. with its attendant step potential and spike recorded by the voltage electrode in the rear region of a moving amoeba. The arrow on the current trace indicates an attempted penetration by means of the current electrode. Coincident with this was a voltage change recorded by the voltage electrode which could be called a positive spike potential. Movement of the voltage-recording electrode then produced characteristic depolarization (Bingley, 1962) and instability, which was followed by characteristic disorganization of the cell contents. The cell swelled and the potential reversed its polarity to give a positive excursion. This has already been described (Bingley, 1962, 1965 b) and will be further described later in this paper.

Figure 7  illustrates the reaction of a pseudopod to prolonged probing in the rear region by the current electrode. An attempted insertion in the rear region of the amoeba is illustrated by means of a dotted line. The current pulses have been removed from the current trace in order to show small spikes which frequently appear when the current electrode is brought into contact with the membrane and thus act as markers to indicate probing. A voltage artifact was associated with this probing on the voltage trace when the voltage electrode was outside the cell; this is marked in the figure. The voltage electrode was then inserted into the pseudopod and recorded a low potential of —19 mV. Further probing in the rear with the current electrode induced a rise of potential in the pseudopod after many seconds. At the same time streaming reversed and the cell burst.

Further study was made on potentials recorded with single microelectrodes whose resistance was monitored throughout the experiment. Special attention was paid to observations made in the rear region of moving A. proteus and to changes in resistance recorded by the electrode while within the cell.

Figure 8  illustrates membrane potentials recorded with a microelectrode whose resistance (R.E.) was 19·0 MΩ in the rear region of a moving amoeba. An initial potential of — 22 mV. was recorded with a drop of the total resistance recorded by the electrode to 17·0 MΩ (this is due to slight breakage of the tip on entering the cell). Further penetration of the microelectrode induced a sudden jump of potential to — 70 mV. with a further drop in the resistance recorded by the electrode to 13·4 MΩ. This value was lower than when the electrode was outside the cell. On withdrawing the electrode very slowly there was a gradual decline in membrane potential with a slight increase of total resistance recorded by the electrode to 14·0 MΩ. On withdrawal from the cell there was an increase of resistance to 16·4 MΩ with a reduction of potential to zero and a return to the base-line.

Figure g illustrates potentials recorded from an advancing pseudopod with a microelectrode whose resistance (R.E.), was 12·4 MΩ. On insertion into the pseudopod a potential of — 45 mV. was recorded with a drop of resistance recorded by the microelectrode to 8·0 MΩ. On removing the microelectrode there was a return to its original resistance value (R.E. = 12·4 MΩ) and a return to the base-line. Of interest is the spike potential appearing on entering the pseudopod. On insertion into the rear region an initial negative spike potential was recorded followed by a short period when the electrode recorded a low potential of — 45 mV. The total resistance recorded by the electrode during this period was 16·4 MΩ. A rapid rise of potential followed, reaching a level of — 80 mV. This was accompanied by a fall in resistance recorded by the electrode to 11·0 MΩ, lower than the resistance of the microelectrode alone (R.E. = 12·4 MΩ). On removal of the microelectrode there was a return to the base-line and an increase of microelectrode resistance to its original value (R.E. = 12·4 MΩ).

If reference is made to Fig. 1 it will be seen that the pulse applied to the microelectrode has a slow overshoot component. For purposes of measurement only the rising phase is used, but Fig. 9 shows this slow component to be attenuated when the microelectrode is inside the membrane.

Figure 10  illustrates a potential of — 40 to — 60 mV. recorded from the rear region of a forward streaming amoeba as shown in the diagram with a microelectrode whose resistance (R.E.), was 2·0 MΩ. A step potential of — 20 mV. was encountered on penetration and the resistance recorded by the electrode increased to 6·4 MΩ. Potentials fluctuated considerably, reaching a peak of — 60 mV. at which point the resistance recorded by the microelectrode was at its maximum (16·4 MΩl). This coincided with a vigorous twitching in the rear region. Thereafter potential and resistance recorded by the electrode fell to zero and 2·0 MΩ respectively. A series of positive potential excursions followed which coincided with the appearance of a small clear area in the vicinity of the tip of the microelectrode (see drawing in Figure 10). The cytoplasmic streaming reversed its direction towards the rear of the cell and the resistance recorded by the electrode fell from 2·0 to 1·5 MΩ and then again to 1·0 MΩ. On removing the electrode from the cell there was a fall in positive potential to zero and the measured resistance (R.E.) of the microelectrode has now fallen to 1·0 MΩ. On this occasion the cell did not burst but new pseudopods were formed in the tail region as shown in the drawing.

These experiments now confirm that low potentials recorded by microelectrodes in pseudopods and high potentials in the rear region are genuine membrane potentials. This experiment is the last in a long series which have already been described elsewhere.

It must be pointed out, however, that experiments involving double electrode penetration are extremely difficult to perform and the sensitivity of A. proteus to two microelectrodes is very much greater than to one. In the majority of experiments it was found that the response to a microelectrode carrying current when there was already a voltage electrode recording membrane potential was rapid, and the form of the amoeba was completely disrupted. Such experiments had to be discarded and only those were accepted in which the cell maintained its original streaming form. It is worth while to make a few comments on this. Normal streaming A. proteus show welldefined tubular pseudopods with hyaline caps whereas A. proteus which have been probed by many microelectrodes frequently assume a less well defined shape; their movement may persist. It does seem that potentials recorded from these latter cells are not the same as those from normal, whole, structured, streaming cells. Workers should take the utmost care when recording potentials from A. proteus so as not to induce the latter form which, although still moving, is basically structureless and can give very misleading results.

Other points of interest have emerged from these experiments. The behaviour of the current-carrying electrode is of interest when it penetrates the cell. In many cases there is an increase of current flowing through the system. Bingley (1964b) showed that microelectrode resistance in saline-filled microelectrodes was progressively decreased when the external ion concentration was increased. The current-carrying electrode will encounter an increase of ion concentration when passing through the membrane and an increase in current flow is what would be expected. The insertion of a current-carrying microelectrode into a pseudopod has a pronounced depolarizing effect on membrane potentials recorded from the rear region, but not vice versa. The obvious implication is that since the current-carrying microelectrode is grounded through a low source-resistance pulse-generator (see Fig. 1) the membrane potential is being shunted down this path; but why is a pseudopod not depolarized under similar conditions? Why in Fig. 2, when the current-carrying electrode is removed from the rear region, is there not a rise of potential? Fig. 4 shows a complete membrane depolarization in the rear region for a membrane resistance value of 2·0 MΩ). In order for this to be a shunting effect the current-carrying electrode would have a resistance at least as low as one-tenth that of the membrane. This yields a value of 200,000 Ω). This microelectrode would have such large dimensions that it would disrupt any amoeba into which it was inserted. This was clearly not so. A similar argument applied to Fig. 5, yields values for current-carrying electrode resistance required to initiate a 50% depolarization of just below that figure which is known to initiate disruptive changes in Amoeba proteus (Bingley, 1964a). These considerations led to a study of those records in which the current electrode failed to penetrate the membrane but would still serve to stimulate it both electrically and mechanically. In this case there should be no shunting effect since the membrane would remain intact in the area of electrode probing.

Fig. 6 shows in its current trace the point at which an attempted insertion was made into the pseudopod by means of a current-carrying electrode. Coincident with this there appeared a positive-going spike on the voltage trace recorded by the voltage microelectrode inserted into the rear region. Numerous other records show similar situations. This prompts the question: is it possible that a stimulus, whether electrical or mechanical, in the vicinity of the pseudopod initiates a propagated potential change travelling to the rear but not the other way round? In other words is there a polarized message-carrying system in Amoeba proteus such that the rear region is immediately electrically aware of events taking place in the vicinity of advancing pseudopods but not the other way round? Again, are these early pointers simply artifacts? Forthcoming experimental work will be designed to answer specifically these questions.

When a microelectrode is slowly inserted into the rear region there is an initial step potential followed by a spike potential of great magnitude which is in turn followed by a steady high membrane potential. It is of interest to speculate how these potential changes are induced by the steady inward movement of the electrode. Fig. 8 and 9 show that there is a decrease of total resistance monitored by the microelectrode during the jump from low to high potential. Since previous work by Bingley (1964b, 1965 a) has shown that electrode resistance is a function of external ion concentration, it is possible that this drop in resistance recorded by the microelectrode, to a value lower than when it was out of the cell, is caused by the sudden production of free ions within the cytoplasm. It is possible that this production of free ions may be associated with the sudden conversion of gel to sol in the rear region. If we assume that this free ion is potassium it is quite possible that this would raise the membrane potential and lower the electrode resistance. If we turn to the detailed structure of the surface of an amoeba and refer to Mast (1926) we find that in the rear region the plasma membrane is separated from the cortical gel by a thin hyaline layer. Goldacre (1952) suggests that contact of the membrane with the cortical gel induces solation and contraction in the latter. It is possible that the act of inserting a microelectrode into the rear region initially depolarizes it. Light prodding in the region adjacent to a microelectrode recording from the rear region induces depolarization (Bingley, 19656). As the electrode is advanced further in, the membrane would then be brought into contact with the cortical gel. It is possible that at this point the spike potential is initiated with the liberation of free ions to restore a high level of membrane potential. Likewise in a pseudopod which is advancing, gentle penetration at the tip produces only low membrane potentials. Deeper penetration leads to step-like jumps in negativity accompanied by reversal of streaming.

Wolfson (1943) divided his membrane potential recordings from Chaos chaos into two categories. Potentials up to — 40 mV. he referred to as the low or A potentials ; high ones, up to —90 mV., he referred to as B potentials. He noted that the B group of potentials were recorded when part of the gel region was sucked into the electrode. Again, was the cortical gel brought into contact with the membrane in the region of the electrode tip?

Such a mechanism as outlined above, if it were real, would be of selective value to an amoeba. The effect of depolarization in the rear due to light touch, and the lack of effect on potential in the pseudopod region, contrasted with the rapid restoration of membrane potential in the rear and its rise in the pseudopod due to deep penetration, would enable an amoeba to distinguish two types of encounter. The first a light touch, probably a food organism, would be sufficient to initiate pseudopods in the rear and not affect them in the front, but a more intense stimulation made by some predator probing more deeply would initiate a spike and rapid movement away from the region stimulated, whether in the pseudopod or the rear. This model is entirely hypothetical but as far as evidence goes it would seem to be of biological advantage to the organism.

The possibility has been advanced that there is a potential difference between the sol-gel interface in amoebae. To explain the potential differences existing between a pseudopod and the rear region on this assumption would necessitate the presence of a resistance barrier between the sol and the gel, otherwise the currents flowing would be excessive. Evidence does not point to the presence of this barrier. When inserting a microelectrode into the rear region there is often a decrease in resistance encountered when there is a step from low to high potential. Neither is there any electron-microscopic evidence of the presence of a second membrane between the sol and the gel regions (E. H. Mercer, personal communication). Workers who encounter step potentials when inserting microelectrodes into rear regions of amoebae should consider the possibility that these potentials are external membrane phenomena.

The observation that the amoeba membrane seems capable of attenuating the lowfrequency component in the pulse applied to the voltage-recording microelectrode is an interesting one since it displays properties which are the opposite to those expected in a membrane, i.e. inductive as opposed to capacitative ones.

The observations made in Fig. 10 illustrate clearly both electrical and morphological changes that take place when a microelectrode breaks when within the cell, The reversal of potential that accompanied the change in the direction of cytoplasmic streaming is shown in the figure as is also the appearance of a clear area around the tip of the microelectrode. The possibility that this may be akin to some very primitive form of action potential has been discussed (Bingley, 1965b).

This work was done under the auspices of a Government Senior Research Fellowship at the R.A.F. 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 thank Prof. H. Holter of the Carlsberg Laboratorium, Copenhagen, Dr J. W. L. 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 are also due to Mr D. R. Baillie of the Photographic Section, Mr C. W. Baker of the Workshops, and Mr S. Hunter, of the R.A.F. Institute of Aviation Medicine.

Lastly I would like to acknowledge the help of Mr Veal of Ernst Leitz Ltd, London, in the initial setting up of the optical and micromanipulative equipment.