J. Cell Sci. 3, 105-114 (1968) 105

Printed in Great Britain

STRUCTURAL ORGANIZATION ASSOCIATED

WITH PSEUDOPOD EXTENSION AND

CONTRACTION DURING CELL LOCOMOTION

IN DIFFLUGIA

A. WOHLMAN1 AND R. D. ALLENf

Department of Biology, Princeton University, Princeton, New Jersey, U.S.A.

SUMMARY

In Difflugia corona, a free-living amoeboid cell, locomotion is hampered by a heavy shell ortest made of sand grains and other debris. Locomotion involves pseudopod extension,attachment to the substratum, and forcible pseudopod retraction which pulls the shelledcell body forward.

When observed through a polarizing microscope, the extending pseudopodia appear iso-tropic or very weakly birefringent. Upon attachment to the substratum a positively birefringentfibrillar array develops rapidly at the attachment point and extends from this region back tothe cell body within the test. These birefringent fibrils extend through and parallel to thelong axis of the pseudopod. As the pseudopod retracts, the birefringent fibrillar array disappears,and hyaline blebs, suggestive of syneresis, appear on the pseudopodial surface. The birefringentfibrils correspond in position and approximate diameter (1 fi) to refractile fibrils visible withthe Nomarski differential interference microscope.

Individual organisms were fixed for electron microscopy at a time when the pseudopodiawere firmly attached to the substratum. Electron-microscopic examination of thin sections ofpseudopodia revealed many i-ft bundles of intimately associated, aligned, 55-75 A micro-filaments. The orientation and size of the bundles indicate that they probably correspond tothe birefringent, refractile fibrils observed in living cells. Microfilaments have also beenobserved both as randomly oriented and dispersed cytoplasmic components, and as alignedfilaments in the ectoplasm adjacent to the plasmalemma.

During pseudopod extension with sporadic streaming, birefringent 'flashes' have beenobserved at the front of the pseudopod. These flashes are believed to represent a photo-elasticphenomenon.

INTRODUCTION

Attempts to analyse the mechanisms underlying amoeboid movement and cyto-plasmic streaming have, for many years, been focused on the search for fibrils orfilaments which, upon contraction, could provide the required motive force. Thesearch has been largely limited to the large, free-living, naked, carnivorous amoebae(see Allen, 1961a). These ideas can be traced back to the early nineteenth century.Following Dujardin's (1835) description of the cytoplasm of amoeboid cells ('sarcode')

• Present address: Bell-Craig Laboratories for Medical Research, 451 Alliance Avenue,Toronto 9, Ontario, Canada.

t Present address: Department of Biological Sciences, State University of New York atAlbany, Albany, New York, U.S.A.

106 A. Wohlman and R. D. Allen

as contractile, and Briicke's (1861, 1862) proposal that the cytoplasm consisted ofa contractile framework, Engelmann (1869, 1875, 1879) further postulated theexistence of 'Inotagmen', uniaxially birefringent, contractile elements capable ofundergoing form changes to produce the force responsible for cytoplasmic movements.

The concept that contractile fibrils or filaments might provide the motive forcefor cell movements and cytoplasmic streaming has also been discussed considerablyin the current literature (for example, see Seifritz, 1929; Meyer, 1929; Monne, 1948;Loewy, 1949; Goldacre & Lorch, 1950; Frey-Wyssling, 1953; and Allen, 1961 b).

The search for fibrils at the light-microscopic level in living cells has not beenvery fruitful; on the other hand, the electron microscope has revealed a number offibrillar elements, ranging between 30 and 80 A in diameter, present in fixed cells(Randall & Jackson, 1958; Sotelo & Trujillo-Cen6z, 1959; De Petris, Karlsbad &Pernis, 1962; Yaqui & Shigenaka, 1963; Danneel, 1964; Nachmias, 1964, 1966;Wohlfarth-Bottermann, 1964a, b; Wolpert, Thompson & O'Neill, 1964; Baker,1965; Komnick & Wohlfarth-Botterman, 1965; McManus & Roth, 1965; Nagai &Rebhun, 1966; Cloney, 1966; Drum & Hopkins, 1966; Masurovsky, Benitez &Murray, 1966; Overton, 1966; Taylor, 1966).

The present paper demonstrates the existence of fibrils in pseudopodia of livingtestaceans and shows that these fibrils form during pseudopod extension, apparentlyto participate in forcible pseudopod retraction during cell locomotion. Closer exami-nation with the light and electron microscopes has enabled us to characterize thefibrils as microfilaments.

MATERIALS AND METHODS

The organism used for this study was Difflugia corona (Wallich), a fresh-watertestacean. The order Testacea, part of the class Sarcodina, contains those amoebaewhich inhabit a single-chambered shell. For general information concerning thetestaceans the reader is referred to the works of Leidy (1879), Mast (1931), Deflandre(1953) and Jepps (1956).

Most of the organisms were obtained from the surface mud or ooze at the bottomof sphagnum bogs in the pine barrens of south-eastern New Jersey. Difflugia coronawas cultured in either the natural waters in which it was found or in Marshall'smedium (0-05 mM MgSO4. 7H2O; o- 5 mM CaCl2; o-15 mM KjHPC^; o-11 mM KH^C^in distilled water), supplemented with diatoms and finely-ground quartz sand. Thecultures were maintained at 18—20 °C under a light—dark cycle consisting of 12 h oflight and 12 h of dark.

Light microscopy involved the use of two contrast-generating systems: polarizedlight and Nomarski differential interference optics (Nomarski, 1955). The opticalcomponents were manufactured by Carl Zeiss (Oberkochen) and were mountedin a Photomicroscope I. Photomicrography on 35-mm film was accomplished bymeans of the internal camera and accompanying automatic exposure device of thePhotomicroscope. Because of the limited light available during observation of theorganism between crossed polars, photographic records were made on Kodak Plus-xfilm processed with Diafine developer which increased the ASA rating 6-9 times

Pseudopods of Difflugia 107

(Baumann Photochemical Corp., Chicago, Illinois). The more favourable lightintensity afforded by ordinary bright-field and differential interference microscopypermitted the use of a finer-grained film, Adox KB-14, which was also developedin Diafine.

Motion pictures were taken with an Arriflex 16-mm camera equipped with eithera synchronous motor drive for 8 frames/sec or variable-speed d.c. motor. Whenframing rates of 2/sec or less were desired, the Arriflex DOM time-lapse apparatuswas used, Kodak Plus-x negative film, developed in Diafine, was used for motionpictures of the organism in polarized light. In the case of bright-field and differentialinterference microscopy, the finer-grained British Kodak High Contrast Pan filmgave the most satisfactory results. Analysis of 16-mm film was accomplished witha modified Kodak 'Analyst' projector ('Photo-optical data analyzer', L-W Photo,Inc., Van Nuys, California).

For electron microscopy, individual organisms were placed in a small drop ofMarshall's medium and allowed to attach to the substratum. Fixation was achievedby flooding with 3*8% glutaraldehyde in 0-13 M phosphate buffer at pH 7-2. After20 min at room temperature, the cell was washed 3 times in Marshall's medium,post-fixed in 1 % osmium tetroxide (1 h), washed again and dehydrated through anethanol series. After the specimen was cleared in propylene oxide it was allowed toequilibrate overnight, at room temperature, in a 1:1 mixture of complete Epon resin(including hardener) and propylene oxide. Following equilibration, the cell wasembedded in Epon (Luft, 1961) and sectioned with a Huxley ultramicrotome.Sections stained with uranyl acetate (Stempak & Ward, 1964) and lead citrate(Reynolds, 1963) were examined in the Hitachi electron microscopes HS-7 andHU-11.

RESULTS

Pattern of pseudopodial movement

Difflugia corona moves by extending cylindrical pseudopodia which attach to thesubstratum, then forcibly retract, pulling the shelled cell body forward. In a typicalsequence in the movement of D. corona, pseudopodia can be seen to extend abovethe substratum, swing to one side or the other, and subsequently settle down andattach. Attachment of the pseudopod to the substratum is quite firm, as evidenced bythe fact that small, membrane-bound fragments are often left on the substratumfollowing pseudopod detachment. These membrane-bound fragments show inde-pendent movement for indefinite periods despite their lack of a nucleus. Numerouspseudopodia may be put forth and the force determining the velocity of locomotionis apparently the vector sum of the forces exerted by the contracting pseudopodia.Competition between pseudopodia extended at approximately 1800 with respect toeach other results in detachment and retraction of the less firmly attached pseudopod,while contraction of the dominant pseudopod determines the direction of the organism.Two adjacent pseudopodia from the same organism will often fuse if they touch atany point along the pseudopodial surface. A cytoplasmic bridge is frequently seen

108 A. Wohlman and R. D. Allen

to form at the proximal end of two adjacent pseudopodia. Membrane fusion hasnever been observed upon contact of pseudopodia from different organisms.

Pseudopod extension and cytoplasmic streaming

Cytoplasmic streaming as observed in D. corona closely resembles that describedfor Amoeba proteus and Chaos caroUnensis. Endoplasm streams but the hyaline capfrequently seen at the front tip of naked amoebae is often not evident in Difflugia.In many instances cytoplasm has been observed to stream into an extending pseudopodand subsequently to continue streaming distally as the pseudopod itself retracts andis withdrawn into the cell body. Consequently, bulbous formations sometimes appearat the pseudopodial tip. In Fig. i the arrow identifies and follows a single particleflowing in the endoplasmic stream while the pseudopod itself is retracting. Reversalof the direction of streaming initiates at the proximal end of the pseudopod (as inC. caroUnensis and A. proteus) and the wave of reversal propagates distally. Cytoplasm,streaming out of a retracting pseudopod, can flow in a U-shaped pattern and enteranother pseudopod without circulating through the cell body. The velocity of streaminghas been observed to range from 2 to at least 15 /i/sec and is highly variable.

Very rapid, brief flashes of relatively strong positive birefringence have been notedto occur in normally weakly birefringent extending pseudopodia undergoing sporadiccytoplasmic streaming. Such flashes are more easily seen in projected films than insingle frames printed from the film.

Fibril formation upon pseudopod attachment

When observed between crossed polaroids, the pseudopods appear very weaklypositively birefringent as they extend from the cell body (Fig. 2 A). Upon attachmentto the substratum, at any point along the pseudopod, strong positive birefringencedevelops rapidly at the attachment area and spreads both toward the test and towardthe front tip of the pseudopod (Fig. 2C—1). In most instances the proximal spreadingof birefringence is more rapid than its distal advance toward the pseudopodial tip.The birefringence does not extend into the hyaline areas that form around theattachment point. As can be seen in Figs. 4 and 5, the birefringence observed inattached pseudopodia can be resolved into an oriented array of birefringent fibrils,each approximately 1 /i in diameter, which extend through the pseudopod and termi-nate in the attachment area. Upon retraction, birefringence typically fades, butsometimes a rapid reversal of contrast was observed just at the end of the retractionphase. Owing to the rapidity of this phenomenon, it is not known whether thereis a reversal in the sign of birefringence.

The fibrillar arrays observed in polarized light are clearly visible as refractile fibrilswhen viewed through the differential interference microscope (Figs. 6-8). The narrowdepth of field characteristic of this optical system at high numerical aperture permitteda determination of fibril location by focusing through the pseudopod. The fibrilsappear to lie in the ectoplasm just beneath the cell membrane. Since both the bire-fringent and the refractile fibrils are evident only after attachment of the pseudopodto the substratum and since they disappear in a similar manner, it is assumed that

Psetidopods of Difflugia 109

they are the same fibrils. In those cases where the plasmalemma appeared folded orrippled, the fibrils did not follow the membrane contour but extended through thepseudopod in a straight course parallel to other fibrils and to the long axis of thepseudopod.

Syneresis

As contraction proceeds, drawing the shelled cell body toward the attachmentpoint, numerous hyaline blebs, suggestive of syneresis, appear on the pseudopodsurface (Fig. 3). Image duplication interference microscopy (Gahm, 1962, 1963) hasshown that the hyaline blebs contain fluid of lower refractive index than the ectoplasmor endoplasm. The blebs are not restricted to any particular region of the pseudopod,but appear all along the surface. The blebs often provide a site for the extension ofnew pseudopodia which display a morphological and physiological pattern indistin-guishable from pseudopodia originating from the cell body. Both types of pseudopodiaare similar in regard to size, shape, cytoplasmic streaming, and contractility.

Behaviour of excised pseudopodia

The locomotor capacity of pseudopodia isolated from the cell body was evaluatedby excising and observing various size fragments. Regardless of how far distallythe separation was made, the isolated fragments continued to move for variableperiods up to several hours. The fragments displayed both cytoplasmic streamingand consistent translatory movement. Unlike the intact organism, small pseudopodialfragments appeared to be attached along most of their length and moved in a mannermore closely resembling the naked amoebae. When larger pieces were obtained byremoving the test and excising pieces of the cell body, the fragments assumed a shapesimilar to the intact organism; that is, pseudopodia extended from a central cyto-plasmic body. Difflugia have been observed to approach and capture an isolated frag-ment and subsequently reassimilate this piece into one of their extended pseudopodia.

Fibril ultrastructure

The birefringence observed in attached pseudopodia by polarized-light microscopysuggested fibrillar organization at the electron-microscopic level.

Electron-microscopic examination of pseudopodia of D. corona revealed 1-/1 bundlesof microfilaments 55-75 A in diameter. Fig. 9 represents a section of the cell bodydemonstrating the nature of the ground cytoplasm and several cytoplasmic compo-nents after fixation by the methods described. A layer of endoplasmic reticulumappeared closely aligned along the cytoplasmic side of the nuclear membrane. Severaltypes of vacuoles were present, some of which appeared empty while others containedelectron-dense material. The mitochondria displayed a pattern of convoluted tubulescommonly found in many protists. On several occasions vacuoles were seen thatcontained a crystalline inclusion: the nature and function of these structures isunknown. Electron micrographs of attached pseudopodia (Fig. 10) demonstratebundles of microfilaments in the ground cytoplasm. Some microfilaments divergefrom the main bundle and can be seen bending around several cytoplasmic components

n o A. Wohlman and R. D. Allen

(Fig. 10, arrow). In this same electron micrograph one can see the numerous vacuolesthat lie adjacent to the micronlament bundle. Although there is a net orientation, themicrofilaments are not strictly parallel to one another. Some areas of microfilamentsappear 'naked' or 'smooth'; however, in most instances particulate material (orperhaps 'side-bridges') seems to be adhering to the filament core. One has thedistinct impression of a periodicity in the arrangement of the particulate material.Microfilaments have also been observed both as randomly oriented and dispersedcytoplasmic components, and as aligned filaments in the ectoplasm adjacent to theplasmalemma (Fig. u ) .

DISCUSSION

Our knowledge of, and viewpoints toward, amoeboid movement have been greatlyinfluenced by the fact that most workers have chosen to study one of the moreabundant giant, naked, free-living, carnivorous amoebae (for example, Amoeba proteusor Chaos carolinensis). Difflugia extends cylindrical pseudopodia in a surprisingly similarmanner, but that is where the similarity ceases. Unlike the afore-mentioned nakedamoebae, Difflugia forcibly retracts its pseudopodia while they are attached to thesubstratum, with the result that the heavy shell is dragged forward.

What makes amoeboid movement in Difflugia especially interesting is that a specialarray of fibrils is rapidly laid down before pseudopod retraction occurs. The formationof this array between the cell body and a forward pseudopodial attachment pointtakes only a few seconds, and the array itself may be more highly birefringent andmore impressive in extent than the mitotic spindle of a marine invertebrate egg.During pseudopod retraction the array fades and disappears.

Forcible pseudopod retraction is not a well-established general feature of amoeboidmovement. Although many have claimed that contraction of the tail is the drivingforce (by hydraulic pressure) for the formation of new pseudopods (see Goldacre,1952), the evidence in favour of this idea is not convincing, and the behaviouralevidence against it is considerable. In the case of Difflugia, forcible pseudopodretraction occurs without any doubt. It is interesting to note, however, that retractionis not necessarily temporally correlated with extension of a new pseudopod, as wouldbe expected if the latter formed as the result of an increased internal hydraulicpressure.

Forcible retraction of pseudopodia seems to be a general feature of many if notall testaceans, especially those with the heavier tests, and probably occurs also insome of the more obscure species of small free-living amoebae. Forcible pseudopodretraction has also been observed in sea-urchin gastrula mesenchyme cells (Gustafson& Wolpert, 1963; Gustafson, 1964). One might guess that this type of cell movementis a more common feature of embryogenesis, histogenesis, and regeneration than iscommonly recognized. Unfortunately, many of the 'working pseudopodia' in embryoshave so far been quite inaccessible to study. Presumably Difflugia serves as animportant model system for the study of this type of pseudopod.

The term ' forcible retraction' implies that something contracts. Since the time ofEngelmann (1879) it has been assumed that contraction can occur only in an aniso-

Pseudopods of Difflugia in

tropic material. Optical anisotropy (especially positive birefringence) has been observedin nearly every structure known or suspected to be contractile (myofibrils, cilia,flagella, reticulopodia, axopodia etc; see Schmidt (1937) for review). Recent studiesfrom this laboratory (Allen, Francis & Nakajima, 1965) established that pseudopodiaof Chaos carolinensis are normally weakly birefringent (B = 2 x io"6), as if theycontained either a very few protein fibrils or consisted of a weakly organized gel.The same is true of newly forming pseudopodia of Difflugia: the weakly positiveaxial birefringence is barely detectable during pseudopod extension except duringsporadic flow, when flashes of birefringence are observed. This pattern of bire-fringence changes is strikingly similar to that reported in Chaos by Allen et al. (1965),and it is tempting to attribute the phenomenon to the same cause in both cases:a build-up in tension between the postulated site of contraction at the pseudopodialtip (Allen, 19616) and a region of resistance more posteriorly located.

The birefringent material that forms into arrays of fibrils just before contractionis particularly noteworthy, for the timing of its appearance in the pseudopod retractioncycle strongly suggests a contractile role. Engelmann, had he found these structures,might have claimed them as his postulated 'Inotagmen', or contractile elements.While the behavioural evidence is suggestive of a contractile role for these fibrils, wecannot prove this from either the light- or the electron-microscopic evidence so far.The diminution of birefringence during pseudopod retraction could be due eitherto destruction or disorientation of the fibrillar material. The electron microscopereveals only seemingly static bundles of 55-75 A microfilaments, without any indica-tion of how these elements might participate in a contractile event. We cannot excludethe possibility that the microfilaments constitute only a part of the contractilemachinery, the rest of which has not been preserved.

The manner in which the fibrillar material appears and certain aspects of pseudopodbehaviour allow us to infer something of the mechanism by which the fibrillar materialis laid down. New pseudopods are almost isotropic, indicating that if the subunitsof the fibrillar material are present, they are randomly oriented. During the latestages in pseudopod extension, just before attachment, pseudopodia typically waveabout, almost like the tentacles of an octopus, before settling on the substratum. Thismay be due to the development of unequal tension along different portions of thenewly formed ectoplasmic tube. An adhesive pseudopod, making contact with thesubstratum, might be subject to internal forces that would normally cause it tobend, but be unable to bend by virtue of its recent attachment to the substratum.This in turn would result in the development of stress in the ectoplasmic tubebetween the attachment point and cell body. Orientation within the ectoplasmictube gel caused by the stress might serve to orient further polymerization of fibrils.It is regrettable that more has not been learned from electron microscopy of thisfibrillar material, its formation and role in contraction. It is hoped that better fixationmedia can be found.

Brief mention deserves to be made of the ability of non-nucleated pseudopodfragments of Difflugia to move in a coordinated and organized manner (see also Kepner& Reynolds, 1923). This is a somewhat different situation from that which exists

i i2 A. Wohlman and R. D. Allen

in A. proteus, where non-nucleate fragments lose the power of successful locomotionwithin a few minutes after enucleation. The retention of the ability of pseudopodfragments to move independently leaves little doubt that the pseudopods themselvescontain the necessary motile machinery.

We are grateful to Professor L. I. Rebhun for many suggestions concerning fixation forelectron microscopy. This work was supported by a research grant from the National Instituteof General Medical Science.

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(Received 26 April 1967—Revised 1 August 1967)8 CellSci. 3

i i4 A. Wohlman and R. D. Allen

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Journal of Cell Science, Vol. 3, No.

Figs. 4, 5. Birefringent fibrils in attached pseudopodia. Zeiss polarized-light optics.This pair of micrographs shows the reversal of contrast at opposite compensatorbias compensation settings.Fig. 6. Refractile fibrils in attached pseudopodia. Nomarski differential interferencecontrast optics.Figs. 7, 8. Refractile fibrils terminating at the attachment area. Fig. 8 shows theorientation of the organism which is suspended from the underside of a coverslip.Nomarski differential interference contrast optics.

A. WOHLMAN AND R. D. ALLEN

Journal of Cell Science, Vol. 3, No. 1

Fig. 9. Electron micrograph showing area within the cell body (c, chromatin; cv,crystal-containing vacuoles; db, dark bodies (probably lipoids); er, endoplasmicreticulum; m, mitochondria; n, nucleus; nm, nuclear membrane; r, ribosomes).x 24000.

A. WOHLMAN AND R. D. ALLEN

Journal of Cell Science, Vol. 3, No. 1

Fig. 10. Aligned, densely packed microfilaments. The arrow points to several micro-filaments bending around cytoplasmic inclusions, x 40000.Fig. 11. Microfilaments (m/) in the ectoplasm just adjacent to the plasmalemma.x 36000.

A. WOHLMAN AND R. D. ALLEN