Specificity of Pseudopodium Induction by the Action of Cations on Amoeba Proteus

J. Cell Sci. 7, 549-555 097°) 549 Printed in Great Britain

SPECIFICITY OF PSEUDOPODIUM INDUCTION BY THE ACTION OF CATIONS ON AMOEBA PROTEUS

J. E. BREWER AND L. G. E. BELL Department of Zoology, University of Southampton, Southampton, England

SUMMARY Some substituted cholines, long-chain aliphatic substituted amines and simple inorganic salts have been tested at different concentrations for their ability to induce pseudopodia from Amoeba proteus. The ability of substituted amines to induce pseudopodia is inversely related to their ability to bind to acid polysaccharides. The reaction of compounds with the surface polysaccharide is probably not in itself the only requirement for pseudopodium induction. A mechanism is proposed in which the properties of the cell membrane are altered by the reaction of compounds with bulky cationic groups with the membrane lipid. The formation of new pseudopodia is not the direct result of a local reduction in the surface charge density or in surface potential.

INTRODUCTION Previous work has shown that local concentrations of organic cations, both complex proteins and simple detergents, will induce pseudopodia from Amoeba proteus (Jeon & Bell, 1965; Seravin, 1968; Brewer & Bell, 1969a). The mechanism of action proposed (Jeon & Bell, 1965; Brewer & Bell, 1969a) involves the reaction of the cations with acidic polysaccharides at the cell surface followed by the passage of the signal so produced across the cell membrane to the cytoplasm. The importance of charge- dependent interactions in the reaction of detergents with A. proteus has been shown by Brewer & Bell (19696), but the results described in the present communication suggest that pseudopodium induction involves greater specificity in the inducing cation. The role of the concentration of the inducing cation has also been investigated.

METHODS Culture methods A. proteus, strain X65 (Ord, 1968), was grown in mass culture in Chalkley's solution by the method of Griffin (i960). Tetrahymena pyriformis was the food organism. Cells were starved (24 h) before use and washed at least 5 times in Chalkley's solution to remove contaminants. Chalkley's solution was of the following final composition: NaCl, 1-37 mM; NaHCO3, 2 2 3 3 4-76 x io~ mM; KC1, 2-68 x io~ mM; Na.2HPO4, 279 x io" mM; CaHPO4, 7-35 x io" mM; MgCl», 4-92 x io~3 mM.

35-2 550 J. E. Brewer and L. G. E. Bell

Chemicals All chemicals were of the best grade available. Succinyl choline (SuccCh), succinyl dicholine (SuccDich), dodecyltrimethylammonium bromide (C]2quat) and dodecyldimethylamine (C12NMe2) were obtained from K and K Laboratories Inc.; dodecanoic acid (CuC00H) and sodium dodecyl sulphate (C12SO4Na) were obtained from BDH Ltd.; phosphoryl choline (PhosCh) was obtained from Koch-Light Laboratories Ltd. and dodecylamine (C]2NH2) was obtained from RN Emanual Ltd. C12NMe2 and C12NH2 were used as the hydrochlorides and C^COOH was used as the sodium salt.

Technique for the demonstration of pseudopodium induction The method of Jeon & Bell (1965) was used. Amoebae were observed in a chamber, depth 1 mm, constructed of pieces of glass slide cemented to a large slide with Araldite. Sufficient Chalkley's solution was present to provide a flat upper surface. Micropipettes, 10 /«n tip diameter, were filled with a solution of the compound to be tested in Agarose (05 %) by compressed air. The tip of the pipette was placed near an amoeba and the reaction of the amoeba was observed and filmed. Induction of pseudopodia, food-cup formation, inhibition of pseudopodium formation and indifference could be clearly distinguished. Com- pounds were retested on at least 2 occasions using at least 10 pipettes. Cinematography at 1 frame/s was used to record the experiments and closed circuit television enabled unhindered observations to be made by persons other than the experimenter.

RESULTS The main groups of compounds investigated were a group of cholines of approxi- mately the same chain length but of different charge ratios, a number of simple inorganic salts and detergents of the general form C12H25R where R was a charged group. The compounds were tested at different concentrations over parts of the range io~4 M-I M. Very high concentrations, e.g. io-1 M, caused non-specific effects irrespective of the pseudopodium-inducing ability of the compounds at lower concentrations. The concentration of cations at the cell surface may be expected to be 2-3 orders of magni- tude lower than the concentration in the pipette (Brewer & Bell, 1969a). The first stage in pseudopodium induction was the cessation of streaming immediately below the cell envelope at a point nearest to the pipette tip (Jeon & Bell, 1965; Brewer & Bell, 1969 a). Further stimulation led to the formation of a pseudopodium in the direction of the pipette. Pipettes containing compounds which did not induce pseudopodia generally had no effect on the direction of formation of pseudopodia by the cell. However, high concentrations of many compounds caused a characteristic non- specific reaction. This took the form of inhibition of streaming in the direction of the pipette and in some cases complete reversal of cytoplasmic streaming occurred and the cell moved rapidly away from the stimulus. Thus, pipettes containing NaCl (1 M in 0-5 % Agarose) positioned in front of an advancing pseudopodium caused a rapid and powerful contraction in the pseudopodium and immediate reversal of the direction of cytoplasmic streaming. Similar effects were obtained with pipettes containing KC1, 1 CaCl2 or MgCl2 at 1 M or C12SO4Na at io" M. This effect is completely non-specific when compared with the effects of these compounds at lower concentrations; NaCl, 3 KC1, CaCl2, MgCl2 and C12SO4Na have no effect in pipettes at io~ M whereas Specificity of pseudopodium induction 551

C12quat induces pseudopodia at this concentration. Similarly the effects of the above concentrations of these compounds on amoebae immersed in them are quite different; at 1 M NaCl and KC1 induce pinocytosis, CaCl2 and MgCl2 are poor inducers of pinocytosis and C12quat and C12SO4Na are lytic. The effect of increasing the concentration of the test compound in the pipettes takes 2 forms depending on whether or not there is a concentration which is effective 4 in inducing pseudopodia. Pipettes containing C12quat at IO~ M and below have no effect on amoebae, which continue to move to extend pseudopodia in directions unrelated to the position of the pipette. At 3 x io~4 M, pseudopodia are induced in nearly all cases but food-cups develop only occasionally. At io~3 M, several pseudopodia are induced and large food-cups are produced about the tip of the pipette. At IO~2M extreme reactions occur in which nearly the whole of the amoeba is engaged in forming an enormous food-cup by the projection of a continuous sheet of the cell towards the pipette. In some cases the cell may be immobilized. At icr1 M immediate reversal of streaming takes place in that part of the cell closest to the pipette and the cell moves away from the stimulus. Pipettes containing NaCl never induce pseudopodia and the change in reaction with increase in NaCl concentration is a change from indifference to avoidance without an intervening attractive concentration. Thus, cells are indifferent to pipettes containing NaCl at io~2 M or below; at io"1 M the reaction is one of avoidance by the inhibition of pseudopodium formation in the general direction of the pipette. Pipettes containing NaCl at 1 M produce a dramatic contraction in approaching pseudopodia followed by the rapid reversal of streaming away from the stimulus. Pipettes containing the following compounds were found to induce pseudopodia in the following order of effectiveness at some appropriate concentration: C12quat > SuccDich > PhosCh > SuccCh > C12NMe2.

C12NH2, C12SO4Na, C^COC-H, NaCl, KC1, CaCl2 and MgCl2 were completely ineffective at any concentration. These results are summarized in Table 1 together with some physical properties of the compounds. The lengths of the unhydrated organic ions were estimated from Courtauld space-filling models; the sizes of the inorganic ions are given in terms of the radii of the hydrated ions. The activity of the compounds was assessed both in terms of the concentrations which elicited pseudopodia and the position along the length of the amoeba from which pseudopodia could be formed (Jeon & Bell, 1965). The change in the activity of the substituted amines with the degree of substitution was striking. The fully substituted compound induced pseudopodia from about two-thirds the length of the cell from the front at io~3 M in the pipette, the rear part of the cell being insensitive, but theunsubstituted primary amine was completely inactive at any concentration. The tertiary amine was only weakly active in the anterior, most sensitive part of the cell and was less active than any of the quaternary amines tested. SuccDich with 2 quaternary amine groups was of about the same activity as C12quat with only one such group but much more active than either PhosCh or SuccCh, which have one quaternary ammonium group together with a negatively charged group (phosphoryl or carboxyl). On t-ri

Table i. Reaction of Amoeba proteus to compounds diffusing from micropipettes

Charge Ionic Size, ratio species 1-1 ' M 3 X IO~4M IO"1 M ^ fcq C12quat i-88 1 :o SuccDich i-8 2:0 - + "T CNMejH i-9 1 :o ° 8 SuccCh i"3 1:1 -I- + PhosCh 1:1 a C12SO4 0:1 CnCOO 1-84 0:1 C12NH2H 177 1 :o Na 0-183 1 :o tq K 0-125 1 :o Mg O-345 2:0 o Ca 0-308 2:0 o + + + +, Extreme food-cup formation; + + +, food-cup formation; ++, pseudopodium induction; +, weak pseudopodium induction; o, no effect; , cytoplasmic contraction; —, inhibition of pseudopodium formation. Specificiy of pseudopodium induction 553

DISCUSSION The control of pseudopodium formation is clearly of great interest in relation to cell movement and cell function. It has previously been shown that pseudopodia and food- cups may be induced from amoebae by local concentrations of proteins (Jeon & Bell, 1965; Seravin, 1968) or quaternary ammonium compounds (Brewer & Bell, 1969a). As the result of his experiments with extracts of hens' eggs, peptone and 7 enzymes, Seravin (1968) concluded that all proteins are capable of inducing pseudopodia. This is clearly not the case, as Jeon & Bell (1965) showed that neither bovine y-globulin nor antibodies directed against the surface coat of amoebae could induce pseudopodia. Similarly, after peptone has been fractionated on an anion-exchange column, only some of the fractions show pseudopodium-inducing activity (J. E. Brewer, 1969, unpublished observations). Since proteins are complex both in their structure and in their chemical properties, we have preferred to work with simpler compounds whose properties are better known. Our discussion of the mode of action of pseudopodium- inducers is therefore confined to considerations of the simple compounds studied by us and does not necessarily account for the action of complex macromolecules. Since the same compound may induce either pseudopodia or food-cups it was suggested that the different responses of amoebae to these compounds arose from differences in the strength of the stimulus (Jeon & Bell, 1965). Thus a strong stimulus was considered to inhibit further extension of pseudopodia; food-cups were then seen to appear when pseudopodia, inhibited at one point, continued to advance on either side where the stimulus was weaker. The present work lends support to this view with the observation that high concentrations of many compounds were inhibitory although pseudopodia could be induced at lower concentrations. The work of Brewer & Bell (19696) suggested that the primary reaction of pseudopodium-inducing cations with amoebae was an ionic reaction between the inducer and the mucopolysaccharide outer layer of the cell envelope. This reaction, together with the concomitant reduction in the negative charge on the cell surface, was considered to be a signal which was passed across the cell membrane to the cytoplasm. On this basis it is to be expected that any compound which reacts with the surface polysaccharide should induce pseudopodia. Long-chain substituted amines react with polysaccharides in the order NH2 > NMe2 > N+Me3 (Scott, 1962), but the order of pseudopodium- inducing ability is N+Me3 > NMe2 > NH2 (no induction). It is apparent that the specificity of the binding sites which leads to pseudopodium induction is different from that of the binding sites on the simple polysaccharides studied by Scott (1962). The acidic polysaccharide nature of the outermost layer of the cell envelope of A. proteus may be demonstrated by metachromatic staining with toluidine blue and there is no reason to doubt that long-chain substituted amines react with this layer of the cell envelope in the same way as they react with the polysaccharides described by Scott (1962). The order of lysis of these compounds for amoebae NH2 > NMe2 > N+Me3 (Brewer & Bell, 1969/;), corresponds to their affinity for acid polysaccharides (Scott, 1962) and suggests that amine-polysaccharide reactions can occur at the surface of amoebae. This being the case it is evident that mere reaction of a compound with the 554 3- E. Brewer and L. G. E. Bell surface polysaccharide is not sufficient for the induction of pseudopodia. The finding that C11COOH and C12SO4Na, which do not react with polysaccharides, do not induce pseudopodia further suggests that, although binding to polysaccharide is not sufficient for pseudopodium induction, it is a necessary step. Our initial reaction mechanism (Brewer & Bell, 1969a) postulated a rearrangement of the lipid part of the cell membrane into a micellar form which would have a lowered transmembrane potential, low electrical resistance and possibly lead to activation of enzymes in the innermost protein layer of the membrane. This arrangement was suggested to be the indirect result of lowering the surface charge of the cell by reaction of cations with the polysaccharide. The results of experiments in which the surface charge was lowered by the presence of locally high concentrations of electrolytes, such as NaCl, show that non-specific lowering of the surface charge is insufficient to initiate pseudopodia. We therefore suggest that the rearrangement of the lipid may be brought about more specifically by the interpolation of quaternary ammonium groups in the plane of the polar head groups of the lipid at the interface between the lipid and the outermost protein layer of the cell membrane. The packing of lipids at interfaces is extremely sensitive to the presence of other moelcules and, in particular, alteration in the spacing of the head groups may lead to large phase changes in the lipid film. Thus, C12SO4Na, C12NH2 and C12quat form liquid filmsa t air-water interfaces; a mixed film of C]2SO4Na and C12NH2 corresponds to a solid phase whereas a mixed film of C12SO4Na and C12quat results in a liquid phase (Leja, 1957). These differences arise from differences in the packing of the polar groups in the interface and the subsequent effect on the van der Waal's forces between the hydrocarbon chains. — NH2 and SO4" groups pack closely together allowing for strong interactions between the chains and the formation of a solid film. —N+Me3 and — SO,f groups can only pack loosely with weaker interchain interactions and the formation of liquid films. If the model of membrane structure proposed by Finean (1953) and Kavanau (1963), in which phosphoryl groups lie in the lipid- protein interface, may be taken as a starting point then it might be expected that compounds altering the packing of the phosphoryl groups would cause phase changes similar to those described above. These phase changes would correspond to changes between the open and closed states of the membrane in the theory of Kavanau (1963). The insertion on additional quaternary ammonium groups into the lipid-protein interface would have profound effects on the packing of the phosphoryl groups with consequent effects on the organization in the lipid layer. If the phosphoryl groups were separated by the introduction of C12quat ions, possibilities for interactions between other parts of the lipid molecules would be reduced as the separation of the chains increased. The change to an open configuration of the membrane follows naturally. We suggest that the specificity of the quaternary ammonium ion arises from its size, positive charge and poor polarizability. The dimethylammonium group is the only other one of the ionogenic groups tested by us which has similar properties and C12NMe2 was the only compound capable of inducing pseudopodia, albeit weakly, which did not contain a quaternary ammonium group. Other effects of the association of inducing ions with the lipid-protein interface include the displacement of other Specificity of pseudopodium induction 555 cations, e.g. Na+ and Ca2+. The importance of changes in the cation composition for the structure of membranes has been emphasized by Kavanau (1963). Reaction with the polysaccharide may thus be seen as a stage leading to the concentration of potentially inducing molecules in the cell envelope; compounds not reacting with the polysaccharide will not be concentrated in this way and compounds not having the appropriate reactive group will not interfere with the packing of the lipid.

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{Received 22 December 1969)