J. Cell Set. 50, 245-258 (1981) 245Printed in Great Britain © Company of Biologists Limited ig8i

FUNCTIONAL INTERDEPENDENCE OFPSEUDOPODIA IN AMOEBA PROTEUSSTIMULATED BY LIGHT-SHADE DIFFERENCE

ANDRZEJ GREBECKI AND WANDA KLOPOCKADepartment of Cell Biology, Nencki Institute of Experimental Biology, ul. Pasteura3, 02 093 Warszawa, Poland

SUMMARYPolytactic cells of Amoeba proteus were exposed to localised photic stimulation. When a

pseudopodium is stimulated to advance, by shading ,it, other pseudopodia are retracted.Activation of the shaded front is the primary response, and contraction of other fronts thesecondary one. When a pseudopodium is inhibited by illuminating its frontal segment, orwhen it is allowed to enter the bright zone in the course of migration, it slows down and stopsbut its eventual retraction depends on the existence of other possible directions for the endo-plasmic flow. Therefore, if other active pseudopodia are lacking, the front suppressed by lightcannot retreat effectively until new fronts arise in other body regions kept in shade. In allexperimental situations the development of new fronts or the activation of forward flow inlateral pseudopodia precedes the contraction of the former leading pseudopodium. Also thereversal of direction of the endoplasmic streaming begins at the new front, and then it graduallyextends until it reaches the former front.

The results confirm the interdependence of different pseudopodia in the same individualand they contradict the concept that pseudopodia behave as separate functional units. On theother hand, they indicate that formation of new pseudopodia should not be explained as asimple secondary effect of contraction of the older ones but, on the contrary, as a phenomenonthat initiates the changes in the pattern of flow in amoeba. The general interpretation is basedon this variant of the pressure-flow theory of amoeboid movement, which attributes the motivepower to the contractile activity of the whole cell cortex and the steering role to events takingplace in the front of the migrating cell.

INTRODUCTION

Analysis of time relationships between the phases of activity of different pseudo-podia in freely moving polytactic amoebae demonstrated that they are correlated(Ktapocka & Gre_becki, 1980). It is not in accord with the assertion that each pseudo-podium ' ...behaves as an independent functional unit' (Allen & Allen, 1978), butit confirms the classical view that 'amoeba acts as an organized unit . . . ' (Mast, 1932).Our earlier experiments allowed us to conclude, in particular, that the moments ofinitiation of new pseudopodia and the moments of inhibition or reversal of movementof older fronts are distinctly correlated in time. However, the methods of analysingthe spontaneous motion of amoeba applied in the preceding study did not permit usto answer the important question as to which of these 2 events takes place first. Isthe formation of a new pseudopodium the necessary condition preceding the retrac-tion of the older one or, on the contrary, is the reversal of functions in the formerlyadvancing pseudopodium the primary factor that initiates a new direction of flow?

246 A. Gr§becki and W. Klopocka

It is possible to promote the development of pseudopodia or to inhibit theirextension by using external factors applied locally to them. Such experiments mayprovide an answer to the question raised above, when they are run with a simultaneousrecording of the effects produced in other, non-stimulated frontal areas. Stimulationby light promises to be particularly convenient in such a study, because it is easy tocontrol its intensity, localization, and time of action. Localized photic stimuli havealready been applied to amoeba in the form of narrow vertical light beams by Mast(1910, 1932) and Schaeffer (1917), and recently by one of us (Gre_becki, 1980) in theform of bright and dark areas projected upon the plane of migration of amoebae,within the field of view of the microscope. This method permits us to induce a newpseudopodium or re-activate an old one by local application of shade, and to inhibitan advancing pseudopodium by local exposure to light.

MATERIAL AND METHODS

Amoebae were grown in Pringsheim medium (Chapman-Andresen, 1958) and fed on Tetra-hymenapyriformis. They were used for experiments 2-4 days after feeding, when they manifest thehighest locomotive activity. Polytactic individuals (terminology of Gre,becki & Gre.becka, 1978)were transferred to a slide, together, with some drops of the original culture medium. Severalglass beads, 0-5 mm in diameter, were added to keep the coverslip at the necessary distancefrom the slide. The slide was then placed, on the stage of the microscope ready for the experi-ment and left for 10-15 IT^n before the beginning of the observations; this allowed the amoebaeto adapt to the new conditions of luminosity and to recover the polytactic mode of locomotion(they became heterotactic during the earlier manipulations). The microscope was equippedwith the differential interference contrast device of the Pluta system (PZO Warsaw). Theexperiments were run in a semi-dark room at 18 ± 2 deg. C.

The light intensity at the plane of cell migration (measured with the photosensitive elementbefore mounting the slide with amoebae on it on the stage) ranged from 6000 to 8000 lux. Itwas white light produced by a standard incandescent lamp with a heat filter. Small rectanglescut out from neutral gelatine filters, sealed between 2 glass slides in order to facilitate manipula-tion, were placed on the top of the lighting apparatus of the microscope, and their image wasreduced by a factor of 10 and focused upon the plane of cell migration using the substagecondenser. The filters used were characterized by 75 % light absorption. The luminosity wastherefore reduced to 1500-2000 lux in the screened parts of the field of view. These 2 levelsof illumination (6000-8000 lux and 1500-2000 lux, respectively) will be referred to in the textsimply as light and shade. In one group of experiments amoebae were allowed to move freelyin light at the beginning, for 10-30 s, and then a single one of their pseudopodia was locallyshaded (by changing the position of the filter in respect to the cell). In other experiments thesame procedure served to expose locally to the light one advancing pseudopodium of a specimenthat was initially migrating entirely in the shade. This method is based on the original idea ofMast (1910) to focus the image of the source of light with the substage condenser, and in thepresent form it was developed by Gre.becki (1980) to study the behaviour of amoebae stimu-lated by a well-defined light-shade difference established across their bodies.

The reactions of the amoebae were filmed with a frequency of 4 frames/s with a 16 mmcamera and time-lapse equipment from Bolex. The films were analysed frame-by-frame withthe LW International Photo-optical Data Analyzer. The successive stages of motion wereredrawn at intervals corresponding to 2-5 or 5 s of real time, and used to plot the activitycurves of all the stimulated and unstimulated pseudopodia produced by an individual in thecourse of the experiment.

Each experiment described in this article has been repeated at least 50 times, and recordedcinematographically at least 30 times. In a few cases (2-4 in each series of experiments) theamoebae failed to react promptly to the photic stimulus. Such experiments were eliminated.The selected examples of behaviour of amoebae analysed in this study represent 80-90 % of

Interdependence of pseudopodia in Amoeba 247

the tests, depending on the type of experiment. It appears obvious, however, that examplesof behaviour different from the dominating pattern must happen sporadically, because anamoeba can ' spontaneously' form a pseudopodium or withdraw it at any moment, which maydistort the picture of its response to the stimulus.

The cinematographic films were also used to produce photokimographically recordedshadowgraphs of the endoplasmic streaming. The method used in producing them was thesame as that used in the study of Kamiya (1950) on protoplasmic flow in the slime mouldPhysarum polycephalum, and those of Rinaldi & Jahn (1963) and Kanno (1965) on endoplasmicflow in amoebae. It consists of projecting the image of the axial part of a pseudopodium (or ofa slime mould channel) through a narrow slit on a continuously running strip of photo-sensitivematerial. The slit must be oriented in parallel to the direction of flow, and at right angles tothe shift of the film strip. The only innovation in the present application of this method con-sists of using cinematographic pictures, instead of recording in vivo the image of endoplaamicstreaming projected directly from the microscope. This makes it possible to produce severalsynchronized shadowgraphs from different body regions of the same individual, by changingthe position of the slit during successive projections of the same film. The equipment used inthis laboratory for the photokimographic analysis of cinematographic records was describedearlier (Cieslawska & Gr?becki, 1978; Gre.becki & Moczon, 1978).

Photographs of the successive stages of motion of amoebae exposed to local stimulationby light or by shade (Figs. 1-6) were taken under the same experimental and optical conditionsas described above for filming.

RESULTS

Activation of a single pseudopodium by shade

Reactions of different pseudopodia of amoebae observed after shading the tip ofone of them are presented in Fig. 1. The serial pictures show the successive phases ofresponse manifested by the shaded pseudopodium, as well as the behaviour ofpseudopodia kept out of the area of stimulation. The gradually changing pattern ofextension and retraction of different fronts of amoeba (as indicated by arrows)strongly suggests that the withdrawal of pseudopodia that remain in light is delayed:it begins when the extension of the shaded pseudopodium is already well advanced.

A more detailed study of this phenomenon was undertaken by means of frame-by-frame analysis of cinematographic records. Series of contour drawings representingthe successive stages of motion at intervals of 5 s were used to construct graphs of theactivity curves of all the pseudopodia analysed. Two such graphs are given as ex-amples in Fig. 7.

Three successive phases may be distinguished in the course of each experiment.The first one represents the period of free locomotion of an unaffected amoeba beforestimulation. The second one corresponds to the lapse of time (5-15 s) between theapplication of shade and the beginning of the visible reaction of the shaded pseudo-podium. In the third phase the pseudopodium exposed to the lower level of lumin-osity reacts vigorously and the effects become manifest in others.

The graph in Fig. 7 A illustrates the sequence of events (similar to that recordedphotographically in Fig. 1) when one of the older pseudopodia, which has alreadybegun to contract, is exposed to shade. This results in the re-activation of its pro-gressive movement, which returns with some delay after the onset of stimulation(15 s in the case described). Then the rapid progression of the shaded front is followed

A. Grgbecki and W. Klopocka

Interdependence of pseudopodia in Amoeba 249

by inhibition and withdrawal of other pseudopodia, which were formerly extended.The stimulated pseudopodium initially behaves as a leading one, but soon it becomesthe only advancing front of the moving cell.

The graph in Fig. 7B shows an example of a different experimental situation, wherethe shade is applied to the tip of one of three well-progressing frontal pseudopodia.The stimulated pseudopodium competes favourably, and it accelerates soon aftershading and takes over the leading role in cell locomotion. The increase in its activityexerts a clear influence on the other fronts. Between 5 and 10 s after acceleration ofthe stimulated front both the other pseudopodia begin to retreat.

In general, the results of frame-by-frame analysis confirm the suggestion based onvisual estimations that the re-activation of forward movement and the intensivegrowth of a shaded contracting pseudopodium (Fig. IA, B) are the first responses tostimulation of amoebae, whereas the retraction of advancing pseudopodia that remainin the bright part of the field comes as a later consequence. The reaction of unstimu-lated pseudopodia is a secondary response to the original reaction of the stimulatedpseudopodium, and not the primary response to the stimulus.

Inhibition of a single pseudopodium by light

Parallel experiments were undertaken to reveal the time relations between the localresponse of an advancing pseudopodium to the increased illumination (its inhibitionand retreat) and the distant effects manifested in other body regions kept outside thefield of stimulation, i.e. in shade. The main objective was to check whether the re-traction of the illuminated pseudopodium is the first reaction of amoebae, and tolearn at which moment the new advancing fronts are formed (or older onesactivated) in unstimulated areas.

Two different experimental situations were studied. In the first the anterior seg-ment of a leading pseudopodium of an amoeba migrating in shade was suddenlyexposed to light by an appropriate movement of the shading filter (Figs. 2, 3). In thesecond the leading pseudopodium was allowed to reach and eventually to cross theshade-light borderline in the course of its own free progressive movement (Figs. 4-6).

Fig. 1. Amoeba stimulated locally by shade after a period of free migration in full light.The shade was applied to a lateral pseudopodium at an early stage of its retraction(A). Note that the stimulated pseudopodium is activated before other fronts begin tocontract (B). At the later stages the stimulated front advances vigorously and othersretreat (C-E). Figs. 1-6, bars, 150 fim; arrows point in the direction of actual endo-plasmic flow; (#) cases of momentary cessation of flow.Fig. 2. Amoeba with a branched pattern of streaming in the frontal zone, at the initialstage of migration in shade (A) ; the moment of exposure of its leading pseudopodiumto light (B) ; retraction of the stimulated front and further growth of the unstimulatedones (C-D).

Fig. 3. Amoeba with a single advancing pseudopodium kept in shade (A) ; the beginningof stimulation of its frontal segment by light (B) ; the temporary cessation of movement(c); the formation and further development of 2 lateral pseudopodia in shade, whichenables the withdrawal of the former front (D-E).

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250 A. Gr§becki and W. Klopocka

Interdependence of pseudopodia in Amoeba 251

In the first as well as in the second variant of the experiment, the amoeba may reactto the increased illumination of its leading front in 2 slightly different ways, dependingon the actual configuration of its frontal body region. An amoeba that had, at themoment of stimulation, more than one actively advancing pseudopodium (Figs. 2 A, B,4A, B) may rather easily and promptly withdraw one of them when it is illuminated.This may be explained by the fact that, in such a case, the flowing mass of endoplasmcan be directed immediately to other actively extending frontal pseudopodia, as isdemonstrated in Figs. 2C, D, 4c, D, E, The cell shown in Fig. 4 presents a particularlygood example of behaviour demonstrating the interdependence of the motion ofdifferent pseudopodia. Each one of its 3 frontal pseudopodia approaches the shade-light borderline and stops; but they approach separately at different moments andthe cessation or the reversal of flow in one of them is always correlated with activationof forward streaming in another.

The second mode of reacting is observed in cells that had not developed any lateralactive pseudopodia at the moment when their front was exposed to light (Fig. 3 A, B)or when it met the shade-light borderline as it moved forward (Figs. 5 A, B, 6A). Inthis case the stimulated pseudopodium continues for a while to progress across theilluminated territory, but soon it slows down and eventually stops (Figs. 3 c, 5 c and6 B). The decrease of frontal velocity after exposing the anterior regions of amoebaeto a higher light intensity was studied quantitatively by one of us and isdescribed elsewhere (Gre_becki, 1981). At the next stage, one or two newpseudopodia are formed at the basal region or along the middle part of the old one(Figs. 3D, 5c, 6c). This means that they always arise with some delay after the partialor complete suppression of activity of the former front, but before its visible con-traction and retreat. The new pseudopodia develop, as a rule, on the shaded part ofthe old pseudopodium (otherwise they are subsequently retracted). Only afterinitiating such new directions for the endoplasmic flow can the illuminated formerfront be effectively withdrawn, as is seen in Figs. 3D, E, 5D, E and 6D.

Activity curves of pseudopodia produced by frame-by-frame analysis demonstratethe same picture. Two examples of these (shown in Fig. 8) show this type of responsewhere amoeba reacts by the formation of new pseudopodia. Again, such an experiment

Fig. 4. Amoeba with 2 equipotential fronts migrating in shade (A); the approach of1 front to the bright part of the field and the division of the other in two (B); theretreat of the first front away from light and the approach of the second one to it (c);the stopping of the second front after crossing the shade-light borderline and furtherextension of the third one (D) ; the cessation of flow in the second and third fronts at thelimits of illuminated zone and re-activation of forward streaming in the first one (E).

Fig. 5. An amoeba approaching the bright field with its unique advancing pseudo-podium (A); crossing the shade-light borderline (B); cessation of further progressacross the light zone and formation of a new front in shade (c); further developmentand extension of the new front and retreat of the old one (D-E).

Fig. 6. Higher magnification of a leading pseudopodium penetrating onto the illumin-ated territory (A) ; cessation of its further progression (B) ; formation of a lateral front inshade (c); its development and retraction of the former one from light (D).

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252 A. Gr§becki and W. Kiopocka

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Fig. 7. Graphs composed of the extension curves of all pseudopodia active beforestimulation, and developed later, in amoebae exposed to the localized action of shade.The beginning of stimulation is indicated by the long vertical arrow. The activitycurve of the shaded pseudopodium is marked by a heavy line for the stimulationperiod. Moments of retraction of other pseudopodia are indicated by smaller arrowsfalling down obliquely. Note that their retreat follows the reactivation of the frontstimulated by shade (A) or the acceleration of its progressive movement (B).

may be divided into 3 distinct phases, similar to those distinguished in the case ofstimulation by shade (cf. Fig. 7). The first phase represents the spontaneous move-ment of non-stimulated amoeba; during the second the illuminated pseudopodiumslows down and stops but never retracts, and in the meantime new pseudopodiaare formed; at the third stage, when new pseudopodia have already developed, theold front, exposed to light, is retracted.

It should be added that in many cases the response of amoeba may represent amixture of both the types of behaviour described above, i.e. the withdrawal of anilluminated leading pseudopodium may be partly facilitated by the presence ofanother active front, and partly dependent on initiation of new pseudopodia.

All the experiments of this group, independently of the manner of application ofthe light stimulus, demonstrate that local inhibition of an illuminated front cannot

Interdependence of pseudopodia in Amoeba 253

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Fig. 8. Diagrams, similar to those shown in Fig. 7, constructed for amoebae locallystimulated by light. The activity curve of the illuminated pseudopodium is markedby a double line for the stimulation period. Moments of formation of new pseudopodiaor of acceleration of the pre-existing ones are indicated by arrows pointing up obliquely.Other symbols are used as in Fig. 7. Note that, as well as after exposing the leadingpseudopodium to light (A), and, after allowing it to enter the bright zone during itsown migration (B), the formation of new pseudopodia (or acceleration of older ones)precedes the definitive retraction of the illuminated front.

lead to the final retraction of the stimulated pseudopodium until new advancingpseudopodia are formed in the unstimulated regions of the cell.

Observations of flow phenomena

Microscopic observations of moving amoeba in vivo, as well as cinematographicrecords analysed either frame-by-frame or by producing shadowgraphs (Fig. 9),allowed us to supplement the data concerning the extension or retraction of wholepseudopodia by adding some information about the mode of changing the directionof endoplasmic flow inside them. The changes of streaming were followed also infreely migrating non-stimulated cells, as in amoebae locally stimulated by light or byshade.

The reversal of endoplasmic streaming in a pseudopodium is most commonlypreceded by a brief cessation of flow. It may often happen as a consequence of illumi-nation of the pseudopodial tip. It seems that in such a situation the streaming stopsalmost simultaneously along the whole length of the pseudopodium (or in the wholeamoeba, in the case of orthotactic individuals). The wave of 'gelation' proceedingfrom the stimulated tip backwards, which has been described by Mast (1932), wasnever observed in the present experiments. After the brief period of rest the streamingstarts in the opposite direction. Usually the reversed streaming is observed first at

254 A. Gr§becki and W. Klopocka

the base of the pseudopodium. Then, the zone of reversal is gradually extended for-wards in a wave-like form, from the proximal segment of the pseudopodium to its tip.In many cases it was evident that, in fact, the wave of reversal started from a newfront formed elsewhere during the period of the inhibition of motion of the olderleading pseudopodium (as described in the preceding section).

A slightly different situation, characterized by the absence of a perceivable phaseof complete cessation of flow, was also quite frequently observed. In such a case, inthe middle and frontal segments of the pseudopodium the endoplasm continues toflow toward the tip (which may still advance), while the streaming has alreadyreversed in its proximal segment. The wave of reversal progresses to the distal end,and it usually takes a few seconds before the uniform new streaming direction is

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Time (s)Fig. 9. Shadowgraph of the endoplasmic streaming in an advancing pseudopodiumthat was initially kept in shade, illuminated between the 25th and 60th second of theexperiment, and then shaded again. The ascendant tendency of streaks produced by themoving endoplasmic granules corresponds to the forward streaming, and their inclina-tion downward corresponds to the reversed direction of flow. Note that the flowreversal during illumination, and its later re-reversal after shading, begin from thebasal region of the pseudopodium (bottom of the shadowgraph), and then extendto the pseudopodial tip (the upper edge of the record).

established along the whole length of the pseudopodium. The observed sequence ofevents is in this case fully consistent with the description given by Allen (1973), basedon the unpublished study of Breuer.

One of the shadowgraphs confirming the gradual character of streaming reversalis shown in Fig. 9. The recorded pseudopodium initially advanced in shade, then theposition of the filter was changed and it was exposed to light, which provoked thereversal of flow and withdrawal of the tip, and finally it was shaded again, whichresulted in the recovery of forward movement. The pattern of the streaks producedby moving endoplasmic granules indicates that in both critical periods (reversal andre-reversal of streaming) the direction of flow changed earlier at the basal part of thepseudopodium than at its more distal regions.

When the anterior segment of a contracting or inactive pseudopodium is exposedto shade the activation of forward streaming begins in the shaded zone, i.e. it startsfrom the pseudopodial tip.

Interdependence of pseudopodia in Amoeba 255

DISCUSSION

The experiments and observations presented in this study allow us to re-evaluatethe reliability of 2 statements made by Allen (1961a, b, 1968, 1973) in support of histheory of frontal zone contraction of amoeboid movement. According to the firststatement, the motor phenomena in different pseudopodia of a polytactic amoebaare unrelated. According to the second, the reversal of endoplasmic streaming alwaysbegins at the tip of a new pseudopodium, and then it is propagated as a wave towardthe former leading front. Both these statements were considered by Rinaldi & Jahn(1963) as contradicting the tail-contraction theory, but qualified as 'incompatiblewith the evidence' and 'erroneously assumed, to support the explanation' given foramoeboid movement by Allen.

The present experiments demonstrate that, in fact, the motor phenomena observedin different pseudopodia in amoebae locally stimulated by light or shade are mutuallyrelated, as was shown previously (Klopocka & Gre_becki, 1980) in the case of spon-taneous locomotion of unaffected cells. The reinforcement of the activity of anadvancing pseudopodium or the re-activation of a contracting one, which are inducedby shading pseudopodial tips, invariably result in the inhibition and retraction ofother formerly active pseudopodia (Figs. 1, 7). The beginning of contraction ofpseudopodia exposed to local illumination is correlated in time with the formation ofnew pseudopodia (Figs. 3, 5, 6, 8), or at least it appears to be dependent on theactivity of other advancing fronts that were present before (Figs. 2, 4).

Therefore, the assumption that pseudopodia are independent (because each one ispulled forwards separately by its own frontal zone) seems to be untenable. The effectsof localized photic stimulation confirm, on the contrary, our earlier conclusion(Klopocka & Gre_becki 1980) that the motor phenomena in different pseudopodia areinterdependent, as might be expected according to the classical concept that endo-plasmic streaming in amoebae follows intracellular pressure gradients.

However, the time relations between the beginning of retraction of older pseudo-podia and the initiation of new ones are hardly compatible with the most commonversion of the pressure-flow theory, which limits the contraction site to the tail ofthe amoeba and to the distal parts of its contracting pseudopodia (e.g. Goldacre &Lorch, 1950; Wehland, Weber, Gawlitta & Stockem, 1979). Rather, it should beexpected, from this last point of view, that the contraction of an old pseudopodiumwould seem to be the original cause inducing the formation of a new one. The presentexperiments demonstrate that in reality the formation of a new pseudopodium beginsearlier, and it is followed (but not preceded) by the effective retraction of the olderfront. When a pseudopodium is stimulated by shade (Figs. 1, 7) it begins to extendforward vigorously before other pseudopodia react. The activation of such pseudo-podia appears not as the consequence but as the necessary condition of withdrawalof other cell parts. When an advancing front is inhibited by light it may slow downor stop, but it effectively retracts only after the development of new pseudopodia atits bases (Figs. 3, 5, 6, 8).

These results are in good agreement with the finding of Seravin (1966), who demon-

256 A. Gr§becki and W. Klopocka

strated that in amoebae immobilized by different physical and chemical shocks theresumption of endoplasmic streaming is initiated by the formation of new pseudo-podial tips. They are also consistent with the observation reported by Kalisz-Nowak(1978), that during the formation of a new pseudopodium the local gap in the ecto-plasmic cylinder appears first, before and not after the change in the streamingpattern in amoeba. This sequence of events is also confirmed by the studies in whichthe withdrawal of older fronts was observed as a further consequence of the experi-mental initiation of a new direction of flow: by sucking the endoplasm with a micro-pipette (Gre.becka, 1980), by perforating the peripheral cell layers (Gr^becka, 1981),by local rupture of the contractile cell cortex by an injected droplet of paraffin oil(Goldacre, 1961; Gre_becka, 1977), by its local disorganization by an injection ofDNase I (Wehland et al. 1979), and by local application of anaesthetics (Korohoda,

1977)-The fact that old pseudopodia begin to retract after the new ones are formed at

their bases makes credible the second assumption of Allen, that the wave of reversalof streaming travels along the pseudopodium from its basal region to the distal end.As a matter of fact, the present observations and records generally support thisparticular statement of Allen (1961 a, 1973) based on the unpublished data of Breuer,rather than the objection of Rinaldi & Jahn (1963). When a new pseudopodium arises,or when an old one is re-activated by shade, the new direction of flow appears firstat its tip. When the former leading pseudopodium is subsequently retracted, the flowusually (but not always) stops simultaneously along its axis, and then the direction ofstreaming becomes gradually reversed from the base of the pseudopodium towardsits tip.

The wave-like propagation of streaming reversal from the new front towards theold one is, in fact, incompatible with the restrictive tail-contraction theories, but itseems to be admissible within the concept of generalized peripheral contraction(Gre_becki, 1979). According to this theory the whole microfilamentous cortex ofamoeba is contractile and creates increased hydrostatic pressure inside the cell. Butthe cortex is discontinuous and dissociated from the outer cell membrane at the tipsof advancing pseudopodia (Gre_becka, 19780,6; Gre,becka & Hrebenda, 1979;Wehland et al. 1979), which lowers the pressure at the cell front and creates hydro-static gradients that promote the endoplasmic streaming. When a new gap opens inthe cortical envelope, as it probably does in the case of stimulation by shade, theoutflow of endoplasm in the new direction will locally reduce the intracellular pres-sure, which may result in the inversion of pressure differences in the vicinity of thenew front, i.e. at the base of the old pseudopodium. It seems feasible that the zoneof the reversed pressure gradient may extend gradually from the new front towardsthe old advancing tip and gradually reverse the direction of flow, as was recorded byBreuer (according to Allen, 1973) and frequently observed by us. The weakening ofthe endoplasmic inflow into the former front creates conditions for the rebuilding ofthe contractile cortex around its tip (as shown by Grqbecka, 19786), which accom-plishes the transformation of the advancing pseudopodium into a contracting one.

In the second situation, when a unique advancing pseudopodium is strongly in-

Interdependence of pseudopodia in Amoeba 257

hibited (as in the case of its local illumination) the rebuilding of cortex around its tipprobably results in raising the intracellular pressure at the front, and consequentlyleads to the disappearance of the pressure gradient and to the temporary cessation ofstreaming. Therefore, the formation of a new front becomes a necessary condition forrestoring pressure differences inside the cell and for the retraction of the inhibitedpseudopodium.

It may be stated in general that the relationships between the formation of newpseudopodia and the retraction of old ones provide no arguments in favour of theconcept that the motive force of endoplasmic streaming is localized in the frontalzone of amoeba. They may be explained adequately by the pressure-flow concept ofamoeboid movement, provided that the theory takes account of the motor functionsof the whole cell cortex (instead of localizing the motive force in the tail region alone)and that it recognizes the steering role of the frontal zone. The mechanism for openingnew gaps in the contractile cortical cell envelope is not yet well understood, but itseems evident that their formation and obturation play an essential role in the frontalcontrol of movement.

This study has been supported by Research Project PAN II.i of the Polish Academy ofScience.

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(Received 10 October 1980 - Revised 19 January 1981)