THE CONTRACTILE BASIS of AMOEBOID MOVEMENT I. The

Home , Amoeboid movement, Cytoplasmic streaming

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT

I . The Chemical Control of Motility in Isolated Cytoplasm

D . L . TAYLOR, J . S . CONDEELIS, P . L . MOORE, and R . D . ALLEN

From the Department of Biological Sciences, State University of New York at Albany, Albany, New York 12203

ABSTRACT

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle . Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca . 7 .0 x 10-' M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations . The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched . When the stabilized cytoplasm was stretched, birefringent fibrils were observed . Thin sections of those fibrils showed thick (150-250 A) and thin (70 A) filaments aligned parallel to the long axis of fibrils visible with the light microscope . Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle . In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane- less pseudopodia to extend into the solution from the .cytoplasmic mass . These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba .

INTRODUCTION

There is now almost general agreement that tion occurring at pseudopod tips has been reviewed amoeboid movement is based on contractility and recently by Allen (1) . Other authors have pre- that the cytoplasm undergoes a contraction in one ferred to reinterpret new data in the light of older region of the cell . There is still some disagreement theories (2-4) . regarding the region that contracts, and there are As in the study of muscle contraction (5), at present only partially substantiated hypotheses progress can be expected to be hastened by the regarding the mechanisms of contractility and availability of model experimental systems less pseudopod formation and how these processes complex than a living intact amoeba . The first may be related . The evidence favoring a contrac- successful attempt at the preservation of motility in

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a preparation of demembranated cytoplasm was showed that Chaos cytoplasm could stream briefly the isolation of "naked cytoplasm" in quartz in a limitied quantity of distilled water between a capillaries (6). Other model systems developed slide and cover glass, presumably because the subsequently have helped to elucidate the mech- cytoplasmic contents were diluted only minimally. anisms of cytoplasmic streaming and contractility Gicquaud and Couillard (16) carried this approach in amoeba. For example, Simard-Duquesne and a step further by designing a physiological solution Couillard showed that glycerinated amoebae in which the motility of A . proteus cytoplasm could could be prepared that would undergo limited be extended for 2-15 min . However, their medium contraction on the addition of ATP and mag- lacked the necessary conditions for the complete nesium ions (7). Amoeba cytoplasm was shown by chemical control of motility. them to possess a calcium-activated ATPase We have now succeeded in isolating cytoplasm activity resembling the ATPase of vertebrate in a lifelike, functional condition from single striated muscle myosin (8) . specimens of Chaos carolinensis by rupturing the Bulk contractility and unorganized streaming plasmalemma mechanically in chemically defined, activity have been observed in preparations of physiological media in which both motility and pooled cytoplasm from Amoeba proteus by Thomp- rheological state can be controlled . The pH, son and Wolpert (9). They also were the first to osmolarity, ionic strength, and K+/Na+ ratio were fractionate pooled cytoplasm and to demonstrate chosen to resemble, as much as possible, conditions the presence of thin filaments in a motile fraction . described in intact amoebae . Pollard and Ito (10) improved the fractionation procedure and demonstrated the presence of MATERIALS AND METHODS both thick and thin filaments in a fraction capable of movement and viscosity changes on the addition Culture of Amoeba of ATP. Motility was depressed by chilling during Specimens of Chaos carolinensis were cultured in -5 isolation and observed only in the presence of Marshall's medium (5 X 10 M MgSO 4, 5 X ATP and on warming to room temperature . How- 10-4 M CaCl2, 1 .47 X 10-4 M K2HPO4, and 1 .1 X ever, the manner of movement in this pooled 10-4 M KH2PO4 in demineralized water) with cytoplasm was different from that in intact mixed ciliates for food . Some specimens were sub- amoebae. Instead of streaming into pseudopodia, jected to centrifugation and bisection with a glass the pooled cytoplasm underwent a massive con- microneedle to reduce the number of light-scattering inclusions traction in which its material shifted in one . Only vigorously moving specimens were used in the experiments reported . direction . I The thick and thin filaments found in motile Solutions extracts of cytoplasm correspond to those observed in electron micrographs of fixed intact amoebae by The solutions used to isolate, stabilize, and control Nachmias (11, 12) . The 70-A filaments have been the motility of amoeba cytoplasm are presented in identified as actin by heavy meromyosin binding Table I . The were based in large part upon studies in Amoeba Proteus (13) and in Chaos carolinensis on the control of contractility in muscle (17-23) . In (14) . The thick filaments have not as yet been addition, they reflect published information regard- characterized biochemically . Although ATPase ing virtually all of the physiological parameters that might be expected to influence cytoplasmic con- activity has been found in amoebae (8), there is tractility . The pH, osmolarity, ionic strength, no evidence that it is associated with the thick potassium/sodium ratio, and divalent cation con- filaments. centrations have been taken into account (24-28) . For some years the need for a more physiological Furthermore, each of these factors was varied experi- cytoplasmic model system has been recognized . mentally over a considerable range to find optimal The original naked cytoplasm preparations (6) conditions for the preservation of distinct contractile exhibited extended viability but were not ideal and rheological states . for physiological experiments because of the Hexokinase solutions were used to study the role of capillary required as a container. Griffin (15) endogenous ATP in contraction . Stabilized cytoplasm (Table I) was treated with test solutions containing various combinations of ATP and divalent 1 T. D . Pollard kindly supplied us with a copy of his cations, both directly and after pretreatment with film of movements in amoeba extracts (1970) . hexokinase solution (0.5 MM MgC12, 5 .0 mM glu-

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 3 79

TABLE I Solutions Used to Isolate, Stabilize, and Control the Motility of Amoeba Cytoplasm

Stabilization Relaxation solution Contraction solution solution Flare solution

pH 7 .0 7.0 7 .0 7.0 mosM 85-100 85-100 85-100 85-100 Ionic strength* (±0 .01) 0 .05 0.05 0 .05 0.05 Pipes buffer mM 5 .0 5.0 5 .0 5.0 Dipotassium EGTA mM 5 .0 5.0 5 .0 5 .0 Disodium ATP mM$ 0 0 1 .0 0.5 KCl mM 27 .0 27.0 27 .0 27 .0 NaCl mM 3 .0 3 .0 3 .0 3.0 CaCl2 MM 0 4.5 0 4 .2 MgC1 2 § 0 0 0 0.5 Free Ca++ < 10 -7 M ca. 1 .0 X 10-6 M < 10 -7 M ca . 7.0 X 10-7 M

* The ionic strength that best preserved the fibrils and consistency in these solutions was 0 .05. However, fibrils that were capable of calcium-elicited contractions were visible in an ionic strength range of 0 .04- 0.12. $ Exogenous ATP is not required for contractions of short duration . Presumably it is bound to the contractile elements and does not diffuse away (see Table II) . Exogenous ATP is required only for prolonged flare streaming . § Exogenous magnesium ions are not essential either for the production of the rheological states or for contractions of short duration (see Table II) . Endogenous magnesium is found in the millimolarity range in amoeba cytoplasm (28) . Exogenous magnesium is required only for prolonged flare streaming .

cose, and 15 .2 U/ml of hexokinase [Sigma Chemical paper strips . When individual cytoplasmic fibrils Co., St . Louis, Mo.]), and both with and without were studied, the solutions were injected into their added 0.5 mM KCN . Control preparations were immediate vicinity with a micropipette . The flow rinsed an equal number of times with stabilization rate of the injected solution was controlled with a solution (Table I) . manometer. Some experiments were performed with the cytoplasm in glass tubes to maintain a greater degree of Isolation of Cytoplasm mechanical stability of the cytoplasm during the At the low calcium concentrations present in perfusion of test solutions . Intact amoebae were stabilization solution, the plasmalemma of Chaos was introduced into the top of a Pasteur pipette (drawn found to become fragile after a few minutes and could out to a diameter of ca. 100 µm) filled with stabiliza- be ruptured with a micropipette 50-100 µm in tion solution . The amoeba was forced through the diameter. The cytoplasm either was allowed to flow length of the pipette by applying pressure to the out of the ruptured cell or was withdrawn with the solution. The amoeba was demembranated as it micropipette by applying a negative pressure of a approached the end of the tapered pipette . The few centimeters of water with a manometer . The pipette was broken around the cytoplasmic plug cytoplasm then slowly spread out over the glass sur- between the isolated cytoplasm and the empty face of the perfusion chamber, which consisted of a plasmalemma . The pipette section containing the slide and coverglass separated by two nontoxic cytoplasm was then placed in the perfusion chamber silicone rubber spacers designed to permit steady and the test solutions were introduced into the per- flow of solutions through the field of the microscope . fusion chamber as above . The cytoplasm adhered strongly to the glass slide and permitted the removal of the empty plas- Light Microscope Observations malemma. In experiments that studied contraction and Measurements and relaxation from the stabilized state, the cytoplasm was oriented with a micropipette controlled The experiments on isolated cytoplasm were ob- by a rack and pinion micromanipulator (Fig . 1) . served with Zeiss strain-free polarized light optics, Solutions in the perfusion chamber were exchanged Nikon rectified polarized light optics, Zeiss Nomarski by introducing test solutions at one end of the differential interference, and by Zeiss Jamin-Lebedeff chamber and drawing them through with filter interference microscopy.

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Experiments in which isolated cytoplasm was caused to contract and/or stream by treatment with the solutions in Table I were observed directly and recorded on films, selected portions of which were analyzed frame by frame with a Vanguard motion analyzer, Vanguard Instrument Co ., Roosevelt, N. Y.). The rates of contraction of fibrils were measured by recording the rate of displacement of particles attached to the fibrils by both frame by frame analysis of selected films and by direct measure- ment with a calibrated ocular micrometer and stop- watch. Viscoelasticity where present was also measured quantitatively as stretch-induced strain birefringence monitored by the same automatic, null-seeking photoelectric birefringence detection system (29) employed by Francis and Allen (30) to observe viscoelastic behavior of amoeba endoplasm in situ . Stabilized cytoplasm was stretched at +45 ° to the axis of transmission of the polarizer. A microbeam of green light (546 nm) was defined by a circular aperture brought to focus in the plane of the cytoplasm. The diameter of the measuring beam at focus was 100 µm, sufficient to illuminate only the cytoplasm. Constant loads were applied to the cytoplasm FIGURE 1 Stabilized state cytoplasm stretched with a through a micropipette using a manometer adjusted micropipette demonstrating fibrils. X 140. to a negative pressure of 6 cm of water. placed in stabilizing solution (Table I) containing Electron Microscopy no exogenous calcium or ATP, amoebae with- draw their pseudopodia, or cytoplasmic streaming Isolated cytoplasm was fixed by perfusing 3% glutaraldehyde solution buffered to pH 7 .0 with 50 is depressed, and the plasmalemma either breaks mM cacodylate buffer . Specimens were postosmicated spontaneously or can be ruptured easily with a 1 h in 1 .0% Os04, pH 7 .0, dehydrated in an ethanol micropipette . The cytoplasm flows slowly out of series, and embedded in Spurr's resin . Cured blocks the ruptured membrane and becomes stabilized were oriented and trimmed in a LKB pyramitome, in a lifelike, viscoelastic condition in which no mo- and thin sectioned on a Reichert OMU-2 ultra- tility is displayed and numerous fibrils can be ob- microtome. Sections were picked up on Formvar- served, especially when the cytoplasm is stretched coated grids, stained in 1 % uranyl acetate, followed (Fig. 1) . The fibrils are characteristically 0 .1-0.4 by a lead citrate stain . The method for negative µm in diameter and are of indefinite length . The staining was based on that of Huxley (31) but cytoplasm remains in this stabilized state for at modified for experiments on single cells (32) . Observations were made with an AEI-EM6B least 10-20 min or until the chemical composition electron microscope with an accelerating voltage of of the solution is altered . 60 kV. A condenser aperture of 250 µm and an When stabilized cytoplasm was stretched, its objective aperture of 50 µm were used . For all birefringence increased (Fig. 2), and upon electronmicrographs the electron microscope was release the cytoplasm recoiled elastically. The calibrated, at the time of use with a no . 1002 cross- viscoelasticity was characterized by monitoring ruled optical grating replica (Ernest F . Fullum, the changes in retardation due to strain bire- Inc., Schenectady, N.Y.). fringence. The birefringence of stabilized cytoplasm increased when tension was applied up to RESULTS the fracture point of the constituent fibrils or when the load was released (Fig. 3) . The increase •in Control of Contractility birefringence under constant load is shown in Stabilization solution, relaxation solution, and Fig. 3, along with discontinuities caused by break- contraction solution (Table I) successfully control age of individual fibrils as they reached their contractility in isolated amoeba cytoplasm . When respective fracture points . Fig . 3 is a continuous

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 381 FIGURE 2 Two polarization micrographs of stabilized cytoplasm being stretched by drawing it into a pipette. (A) positive compensation, and (B) negative compensation micrographs photographed a few seconds apart . The retardation is ca . +20 ,k with the slow axis parallel to the fibrils . (S) specimen, (P) plane of polarizer, (AN) plane of analyzer . X 185. pen recording which is representative of many Chaos endoplasm as it becomes everted to form the experiments . When the cytoplasm was unloaded ectoplasmic tube (33, 34) . the retardation did not return to the original The presence of free calcium ions is required for value, but it exhibited a residual positive retarda- contraction, whereas other free cations at phys- tion (positive deformation) . iological concentrations do not elicit contractions . The contraction solution differs from stabiliza- Contractions were also observed in freshly pre- tion solution principally in its calcium concentra- pared stabilized cytoplasm on the addition of a tion . When applied to stretched stabilized cyto- simple calcium salt solution in the absence of plasm, it produced directional contractions of the exogenous magnesium and ATP . When calcium visible fibrils. The diameters of these fibrils in- buffer solutions with free calcium ion concentra- increased as they shortened to a small fraction of tions between 10-8 and 10-5 M were applied to their original length . These contracting fibrils stabilized cytoplasm, the threshold for contraction developed tension, since portions of broken glass was found to be ca . 7.0 X 10-7 M. The rate of rods attached to the fibrils were transported during contraction increased with calcium concentration contractions. In an unoriented mass of stabilized as shown in Fig. 4 . The time-course of contraction cytoplasm, where distinct fibrils were usually not of a single fibril contracting toward its point of seen, isodiametric contraction occurred as in glyc- attachment to the glass slide in the presence of erinated amoeba models (7). 1.0 X 10-3 M free calcium ion is shown in Fig. 5 . The refractive index of the cytoplasm under- The rate of contraction decreased noticeably as went an obvious increase during contraction . This the fibril shortened beyond one-half of its rest change was comparable to that observed in intact length.

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Time (seconds)

FIGURE 3 Photoelectrically measured changes in retardation because of birefringence on stretching stabilized cytoplasm by drawing it into a micropipette under constant negative pressure (6 cm of H20) . The increased birefringence fell off sharply when the deforming force was removed, but some birefringence remained . When the same experiment was performed after washing with relaxing solution, no increase in birefringence was observed. The decrease observed is because of a diminishing amount of oriented material as it is pulled apart . (T) gamma retardation .

35 70

60 N 30

E 50 25 d 40 0 20 N N 0 30 C Y O u 20 °7 10 & 0 10 0 5 N 0 1 1 I I Q 1 0 4 0- x-ec X Xo A o e e e 0 1 2 3 5 1 I I I I 1 1 1 Time (seconds) 8 7 6 5 4 3 2 I FIGURE 5 Time-course of shortening as percent of the -log lo [cation] resting length of a single fibril contracting in the pres-

-3 . The average rate FIGURE 4 Average velocity of contraction in a popula- ence of 1 .0 X 10 M free calcium ion tion of fibrils . The data were obtained using frame by of contraction of this fibril is 30 .5 µm/s as described in frame cine analysis of the velocity of displacement of Fig. 4 . individual particles attached to the fibrils . (X-X) calcium, (0-0) magnesium, (h-A) potassium and so- Table I was unnecessary for the relaxation of dium . amoeba cytoplasm . Thus, for simplicity, the relaxation solution without magnesium salt (Table Stabilized cytoplasm treated with relaxation I) was adopted as the standard relaxation solution solution gradually lost its viscoelasticity and bire- for all the experiments reported in this paper. fringence (Fig . 3) . The visible fibrils became very The ionic strength that best preserved the fibrils plastic and neither cohered nor fractured when and consistency in these solutions was 0.05 . How- drawn out . The relaxed state could be reversed by ever, functional cytoplasm was observed in an the addition of stabilizing solution . The addition ionic strength range of 0 .04-0.12 . of magnesium salt to the relaxation solution in The visible fibrils could be caused to contract,

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 383

relax, and contract again by carefully injecting Electron Microscopy the required solutions with a micropipette into The ultrastructural basis for the viscoelastic the vicinity of the cytoplasm . Successful cycling properties of stabilized cytoplasm has been required the use of minimal calcium concentrations explored both by negative staining procedures (32) ; otherwise, the fibrils contracted (ca . 7 X 10 -1 M) and by thin sectioning of oriented fibrils in cyto- . rapidly and irreversibly plasm isolated from single amoebae (Fig . 6) . The The isolated cytoplasm could also be converted fibrils visible in the light microscope consist of from one state to another inside a glass micro- bundles of numerous 70-A thin filaments with pipette . If the micropipette was placed in the 150-250-A thick filaments and a few smaller, perfusion chamber and the solutions were grad- uncharacterized ca. 45-A filaments (Fig . 6 a and ually exchanged, the mechanical and chemical b) . integrity of the preparation were preserved to a At the ultrastructural level, relaxation solution greater extent than in naked cytoplasm in the per- caused separation of the filaments and permitted fusion chamber. In the pipette, cyclic changes of a more detailed view of the thick filaments (Fig . state could be performed for up to 4 h at room 6 c) . In higher resolution, negatively stained electron microscope images, the morphology of temperature . the thick filaments from Chaos was found to be When stabilized cytoplasm was incubated in identical with that of myosin aggregates from the hexokinase solution for 5 min, the rates of vertebrate striated muscle and from Physarum contraction were shown to be dependent not only (35, 36) . Upon relaxation, the cytoplasmic fibrils on calcium concentration (Fig . 4), but on the dissociated into individual myosin aggregates presence of magnesium and ATP . Calcium, mag- and actin filaments (Fig . 6 c) . nesium, and ATP caused contractions eight times more rapid than calcium and ATP alone (Table Naked Pseudopodium Formation II) . The values in Table II are the average of 10 and Locomotion experiments . The "flare solution" containing ATP, magnesium ions, and threshold calcium caused naked TABLE II pseudopodia to extend from isolated cytoplasm

Average velocity of con- (Fig. 7) . In flare solution, freshly isolated cyto- traction µm/s plasm retained its ability to stream and form

Stabilized cy- pseudopodium-like structures . There was a direct Untreated toplasm after correlation between the motility of isolated cyto- stabilized hexokinase plasm in flare solution and the motility of the Test solution cytoplasm treatment intact cell from which the cytoplasm was isolated . The most active preparations were always obtained 0.5 mM Ca++ 23 .0 5 .5 from the most actively moving amoebae, regard- 0 .5 mM ATP 0 0 less of their nutritional state . Neither the exogenous 0 .5 mM Ca++ + 0 .5 mM 20.0 3 .0 ATP magnesium nor the exogenous ATP in the flare 0.5 mM Mg++ 0 0 solution was required for short-term streaming . 0.5mMMg+++0 .5mM 0 0 However, the combination of magnesium and ATP ATP was found to increase the duration of motility 0.5mMCa++-h0 .5mM 22 .0 25.0 by a factor of 10 . In flare solution, the peripheral Mg++ + 0 .5 mM ATP layer of cytoplasm came into contact with the solution and a contraction was elicited in such a Stabilized cytoplasm was treated with test solutions way that loop-shaped pseudopodia up to several containing various combinations of ATP and di- hundred micrometers in length erupted from the valent cations, both directly and after pretreat- surface of the cytoplasm (Fig: 7). The local ment with standard hexokinase solution (0 .5 mM MgC12 , 5 .0 mM glucose, and 15 .2 U/m1 of hexoki- application of flare solution from a micropipette nase) and both with and without added 0 .5 mM at a restricted area of stabilized cytoplasm also KCN which did not affect the results . Velocity of caused localized flare formation . Similarly, cyto- contraction was measured as in Fig . 4 . palsm could be transferred to flare solution from

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FIGURE 6 Longitudinal sections of stabilized state fibrils demonstrating : (A) The approximately parallel arrangement of the ca. 70 A thin filaments within the fibril . X 65,110 . (B) The presence of thick filaments (arrow) within the fibril . X 65,100 . (C) This electronmicrograph shows a negatively stained myosin-like aggregate seen after relaxing stabilized state cytoplasm . The aggregate is ca . 0 .5 ym long, has a bare cen- tral region 0 .1-0.2 µm long, and irregular projections at either end . X 165,000 .

stabilizing solution and exhibit flare pseudopod differing degrees of asymmetry to its extreme, a formation . loop pattern (6). Previous studies on isolated cyto- The pattern of streaming in intact pseudopodia plasm have demonstrated the breakdown of the varies from a symmetrical fountain pattern through fountain pattern into loops (6). Isolated cytoplasm

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 385 FIGURE 7 A naked cytoplasmic droplet from the giant amoeba, Chaos carolinensis, in flare solution from which pseudopodia extend into the solution. X 170 . in flare solution more often streams in the loop µm/s (range 19-43 gm/s). When the cytoplasm pattern, presumably because the cytoplasm is not reached the tip of the flare, it contracted on turn- confined (Fig . 8) . ing, underwent an increase in refractive index, and At the concentration of free calcium given in then either remained stationary or migrated back Table I for the flare solution, the velocity of out- into the droplet much less rapidly (0-12 µm/s) . ward cytoplasmic streaming averaged ca. 30 Such "flare streaming" persisted for up to 10 min.

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FIGURE 8 A loop streaming in a plane parallel to the glass substratum in flare solution . These two micrographs (A and B) are of the same loop taken a few seconds apart . The cytoplasm which is capable of sup- porting tension is initially pulled out by a contraction at the bend where the cytoplasm thickens and increases in refractive index. X 540.

Although ATP is required for long-term stream- The concentrations of calcium and magnesium in ing, changing the ATP concentration over a range the cytoplasm are not precisely known . However, of 0 .1 mM-1.0 mM had no effect on the velocity the results of experiments showed a dependence of streaming. of the duration of motility on the concentrations Locomotion of isolated cytoplasm often occurred of calcium, magnesium, and ATP . when the returning cytoplasm in a flare-streaming The absence of an external membrane surround- loop attached temporarily to the substratum . The ing the cytoplasm in the stabilized, relaxed, and extent of locomotion was often several millimeters flare states has been established with confidence on over a few minutes . the basis of the following observations . Neither a If the free calcium concentration was increased steep gradient in optical path nor a birefringent within a factor of two, the rate of streaming in- surface could be detected under optical conditions creased dramatically but lasted for a shorter time . adequate for the detection of unit membrane . In Above 1 .0 X 10-s M free calcium, the naked addition, not only could cytoplasmic "threads" pseudopodia contracted into the cytoplasmic mass . be drawn away from the fabric of the isolated The volume of cytoplasm in these experiments cytoplasm with a micropipette (Fig . 1), but the was always less than 1 /1000th that of the solution . dye eosin Y penetrated the cytoplasmic mass

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 3 87

within seconds while minutes were required for brate striated muscle . On passing from rigor to penetration into intact amoebae . the relaxed state, muscle becomes highly extensible because of the loss of cross-linking between actin DISCUSSION and myosin . That a similar transition occurs in amoeba cytoplasm is demonstrated in Fig Advantages of Isolated Cytoplasm . 3. There is an increase in retardation in the stabilized as a Model System cytoplasm under a constant load and an in- Isolated cytoplasm prepared by the present stantaneous decrease in retardation when the load method has several advantages over other model is removed . These viscoelastic properties of systems : (a) No extreme mechanical or chemical stabilized (rigor) cytoplasm can be accounted pretreatment (such as centrifugation, glycerina- for by crossbridges between the actin and myosin filaments tion, or chemical skinning by detergents or en- . This argument is supported by the loss zymes) is required ; (b) the chemical environment viscoelasticity of the cytoplasm in the presence of can be controlled in order to investigate the condi- relaxation solution which should break cross tions necessary for contractility and streaming ; bridges between actin and myosin (Fig. 3) . (c) the physical integrity and, to some extent, the Marked changes in rheological properties such rheological consistency of cytoplasm are main- as are observed in the cytoplasm of amoebae could tained ; (d) contractility and the ability to form conceivably be caused by the polymerization and organized pseudopodial extensions are preserved . depolymerization of either or both types of fila- A qualitative indication of the success of this ments as was inferred from the viscometric ex- approach is that high quality preservation of periments of Pollard and Ito (10) on pooled cytoplasmic organelles is achieved in both light amoeba cytoplasm . However, a more likely ex- and electron micrographs. planation for cyclic rheological changes in the The stabilization solution (Table I) constitutes intact cell would be control exercised by endog- a "rigor solution" for amoeba cytoplasm because enous free calcium ions and ATP on the interac- it lacks ATP and free calcium ions . Furthermore, it tion of actin and myosin in the intact cell. is buffered to maintain the free calcium con- The evidence that contraction is calcium de- centration below the threshold concentration for pendent and that the rate of contraction is propor- contraction . tional to the calcium concentration makes cal- The contraction solution differs from stabiliza- cium the most probable control ion . Vesicles be- tion solution principally in the presence of free lieved to constitute part of a calcium sequestering calcium ions (ca . 10- s M) . The increase in ionic system have recently been described (38) . strength because of added calcium is insignificant, The observed increase in the rate of contraction as shown by control experiments with added of fibrils with increase in calcium concentration monovalent cations . Short-term contraction does is thought to be because of the increase in the not require exogenous ATP. The availability of number of calcium-activated filaments interacting . endogenous ATP in isolated cytoplasm is sug- Thus, a saturation for calcium activation is not gested by the results of experiments with hexo- observed, since calcium ions are not delivered to kinase . The relaxation solution differs from every contractile unit simultaneously as in ver- stabilization solution principally by the presence tebrate striated muscle . of 1 mM disodium ATP. The rapid decrease in the shortening rate of the fibril during the last one-half of the contraction described in Fig Cytoplasmic Contractility . 5 is believed to be because of the compression and disorientation of the filaments . It is now apparent that isolated amoeba cyto- This disorientation may be analogous to the "super- plasm can be induced to exist in rigor (stabilized), contraction" of muscle . The inability to relax relaxed, and contracted states by solutions similar fibrils contracted into a mass supports this sugges- to those used to control contractility in vertebrate tion . striated muscle (17, 18, 37) . The contraction of stabilized cytoplasm with the The rigor (stabilized) and relaxed states can be addition of buffered calcium salt solutions in- distinguished in amoeba cytoplasm by criteria dicates that an energy source and any necessary similar to those employed in the study of verte- cofactors for contraction remained available to the

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contractile elements . The hexokinase experiments the bend (Fig . 8) . The site of change of refractive shown in Table II suggest the requirement of both index and the ratio of streaming velocities in the magnesium and ATP for maximum rates of con- flare streaming model are identical to the measure- traction. These results indicate that both ATP ments made on intact cells (33, 34) . Therefore, and magnesium ions are contained in freshly iso- the flare loops are believed to represent a com- lated stabilized cytoplasm . Since the volume of ponent of the "fountain" pattern described by the isolated cytoplasm is about 1 / 1000th that of the Allen (42) . solution, it is reasonable to assume that these sub- Flare streaming can be prolonged only by the stances may be bound to some elements in the addition of both ATP and magnesium ions . In- cytoplasm . creasing the ATP concentration over a range of 0.1 mM-1 .0 mM had no effect on the velocity or Ultrastructure mode of streaming . However, since the ATP and mangesium ion concentrations cannot be con- The identity of the thin filaments as actin or trolled rigorously in these whole cytoplasm prepa- actin-like has already been indicated by the heavy rations, the complete understanding of the role of meromyosin binding studies of Pollard and Korn ATP and magnesium ions awaits further bio- (13) for Amoeba proteus and of Comly (14) for chemical analysis . . The thick filaments have remained uniden- Chaos When the free calcium ion concentration is in- tified from electron micrographs of thin sections creased above 10-6 M, the naked flare pseu- although several authors have speculated that they dopodia contract rapidly into the cytoplasmic might be myosin . The negative staining results in- mass . This result is expected if enough free calcium dicate the presence of myosin aggregates . While ions are suddenly made available to activate a positive identification of these structures as myosin large portion of the contractile elements in the aggregates requires biochemical confirmation, it is cytoplasm . interesting to note that Chaos is the first cellular The fact that different types of motility can be primitive motile system in which such presumptive induced in isolated amoeba cytoplasm merely by myosin aggregates have been documented . manipulating the free calcium ion concentration suggests the possibility that different modes of Site of Application of the Motive Force locomotion in related primitive motile systems The details of streaming in flares permit us to might be attributable to differences in the control draw conclusions about how the motive force for system which varies the free calcium ion concentra- this movement is applied . The flare solution was tion . For example, rapid pseudopod retraction, designed to test the prediction that localized con- which occurs infrequently on strong mechanical or traction in a viscoelastic cytoplasm would result in electrical stimulation in Chaos, could be because of stable patterns of cytoplasmic streaming . massive release of calcium from storage in vesicular Because the cytoplasm does not flow out through elements (38) . Similarly, forcible contraction of any kind of tube or other confining structure, the pseudopodia in Dfugia might be because of a flow could not conceivably be caused by a pressure calcium control system capable of releasing gradient . Because the "arms" of the cytoplasmic amounts of calcium sufficient to cause a forcible loops are often not contiguous, the force cannot be contraction of a highly organized bundle of con- generated by any lateral interaction (or "active tractile elements (43) . Eckert2 has demonstrated shear") between the outward and inward flowing the presence of numerous vesicles along the en- masses . Therefore, the type of active shearing tire length of the microfilament bundles believed to postulated to operate in Nitella (39) or Allogromia be responsible for rapid contraction of Dif lugia (40) could not account for pseudopod extension pseudopodia. and streaming in Chaos. These results appear to The velocity of streaming in intact cells is invalidate the recent suggestion of an active shear- variable over a considerable range and very sen- ing model for amoeboid movement (41) . sitive to changes in environmental conditions . It is The ratio of streaming velocities in the outward and inward streaming arms of the loops and the 2 B . Eckert . 1973. Pattern organization of thick and increase in refractive index at the tips of the loops thin microfilaments in contracting pseudopodia of demonstrate that the site of contraction occurs at D fugia. In preparation .

TAYLOR, CONDEELIS, MOORE, AND ALLEN Contractile Basis of Amoeboid Movement I . 38 9 now possible to infer that at least two factors play the substratum on the other that provides this a role in determining the velocity of streaming . One anchor . Isolated cytoplasm can show amoeboid is the rate of shortening of the cytoplasm and the locomotion only when the returning cytoplasm other is the degree of shortening . From our results (ectoplasm) of a loop adheres directly to the slide . it appears that free calcium ions affect both of these variables . A likely third factor is viscous or Question of the Reality of viscoelastic resistance in which the concentration Amoeba Fibrils of ATP very likely also plays a role . The idea that amoeboid movement is caused by Role of the Plasmalemma the action of cytoplasmic fibrils can be traced back to observations on fixed cells during the 19th The plasmalemma with its attached glycocalyx century (cf, de Bruyn [48] for review) . The ques- has long been recognized to represent both the tion of reality versus artifact has always been permeability barrier of the amoeba and the struc- present wherever fixation or experimental manipu- ture by means of which the cell normally attaches lation is involved in determinations of cytoplasmic to its substratum . The plasmalemma has also been structure . In the present experiments on stabilized postulated to play a role in supposed ectoplasmic cytoplasm the fibrils are sometimes visible as contraction (44), in generating a gradient in bio- soon as the cytoplasm pours out of the broken electric potential that might cause streaming (45), cell membrane . They are made more easily and in controlling movement through local sur- visible in cytoplasm oriented by stretching . This face charges (46) . does not necessarily mean that they are not It is now evident that pseudopodia can form in artifacts, for either the stabilizing solution and/or the absence of a plasmalemma or other limiting mechanical deformation might cause their forma- membrane . Furthermore, cytoplasmic contraction by lateral aggregation, under stress, of pre- tion and relaxation can be induced by controlling viously oriented submicroscopic linear elements the calcium and ATP concentration without the (actin and myosin) . It is tempting to suggest that presence of a limiting membrane . Thus, whatever factors internal to the amoeba may control the ATPase may be involved in motility does not degree of lateral interaction of linear elements from reside in the plasmalemma . It is more likely that a state observable as diffuse birefringence to a the thick filaments are myosin and that ATPase condition in which discrete fibrils may be seen . activity resides there . However, this remains to be Such factors as local divalent cation concentration demonstrated biochemically . and mechanical stress might control such interac- It has long been known that a "surface precipita- tions. Nakajima and Allen (49) observed such tion reaction" occurs whenever naked cytoplasm differing degrees of lateral aggregation in the comes into contact with a medium containing plasmodial cytoplasm of the acellular slime mold, free calcium ions (47) . We have observed such a Physarum polycephalum . Wohlfarth-Botterman (50) reaction in experiments in which calcium salts are found an increase in the number of cytoplasmic added to stabilized cytoplasm . However, the fibrils when plasmodial strands were hung so that membrane formation observed was always in- streaming had to overcome gravitational accelera- complete and neither interfered with nor was tion . Kamiya, Allen, and Zeh (51) have more necessary for contraction . recently observed that moderate mechanical While much remains to be learned about the stretching of plasmodial strands increases the force role of the plasmalemma in amoeboid movement, they can develop on contraction . These data are it appears not to be required for cytoplasmic consistent with the hypothesis that mechanical streaming into naked flare pseudopodia which stress may increase both the efficiency of contrac- are frequently deployed in a manner remarkably tion and the degree of lateral aggregation under similar to the deployment of intact pseudopodia . in vivo conditions . Thus, some of the suggested roles for the plasmalemma can probably be discarded. One important Theory of Amoeboid Movement role must be emphasized : amoeboid locomotion can occur only if the ectoplasm is anchored to the It has long been known that the cytoplasm of an substratum . It is local attachment of the plasma- amoeba circulates in a fountain pattern through lemma to the ectoplasm on the one hand and to the inner and outer regions of the cell (48) . Mast

390 THE JOURNAL OF CELL BIOLOGY • VOLUME 59, 1973 Attachment zone

FIGURE 9 A schematic diagram of processes in amoeboid locomotion . Velocity (V) profiles show the patterns of plasmalemma (PL) displacement and cytoplasmic streaming in the front, middle, and rear regions . The region of ectoplasm-plasmalemma-substrate attachment is indicated . The relationship among the region of frontal contraction, the hyaline cap (HC), and plasmagel sheet (PGS) are shown at the pseue dopod tip. Regions of the cytoplasm corresponding qualitatively with the rheological states of isolated cytoplasm are labelled SS (stabilized state), CS (contracted state), CS---SRS (transition from contracted state to relaxed state), and RS (relaxed state) . (HE) hyalin ectoplasm, (GE) granular ectoplasm .

(52), in his classical ectoplasmic contraction "sol- plasm. Such a change probably represents the gel" theory of amoeboid movement, incorporated "gelation" postulated to occur as the endoplasm the well-known fact that the consistency of the becomes everted to form the ectoplasmic tube . cytoplasm underwent changes in different regions The cytoplasm of the amoeba circulates around of the cell . Although these changes have long been the cell in a fountain pattern} as the latter ad- described in oversimplified terms as a "sol-gel vances in relation to its substratum . The ecto- transformation," there is no doubt that the inner plasm in the posterior region of the cell "relaxes", region of the cell, the endoplasm, has a "softer," analogous to the loss of viscoelasticity in relaxed more compliant consistency . However, even the stabilized cytoplasm (Fig. 3), then forms the ma- endoplasm has been shown to possess viscoelasticity terial from which the endoplasmic stream is (30), and therefore it is not correct to refer to it as recruited. The relaxation is probably accounted a "sol ." for by assuming that the cell has a mechanism for The results of the present experiments offer sequestering calcium and controlling the local useful insights into the possible significance of the concentration of endogenous ATP . The tail endo- rheological changes in intact amoebae that Mast plasm has a weaker birefringence than that of the (52) referred to as sol-gel transformations . The anterior cytoplasm (53), suggesting that its degree cytoplasm streaming into pseudopodia is known of actin-myosin interaction is less . If this were not to be viscoelastic ; therefore, it is qualitatively so, then the tension in the endoplasm would in- similar to stabilized, isolated cytoplasm . The crease until its fracture point was reached, and presence of viscoelasticity is significant because it steady-state pseudopodial extension could not allows tension from frontal contraction to be occur . transmitted along the endoplasm backward toward Although free calcium ions have been shown to the tail. control contractility in amoeba cytoplasm, it is Release of free calcium in the pseudopodial tip not known whether exogenous free calcium may region presumably causes contraction in the region enter pseudopod tips from the environment or where the endoplasm becomes everted to form the whether endogenous calcium is sequestered and ectoplasmic tube . As the cytoplasm contracts, it released by organelles. squeezes out some of its interstitial water into the The fact that isolated cytoplasm can be made to hyaline cap and increases in refractive index (33, cycle repeatedly through stabilized, contracted, 34) . Presumably, contraction also results in an and relaxed states suggests that the rheological increase in rigidity as in muscle and isolated cyto- cycle in the intact amoeba during locomotion may

TAYLOR, CONDEELIS, MOORE, AND ALLEN, Contractile Basis of A?rnoeboid Movement 1. 391

be similar. Fig . 9 represents an attempt to depict gradient theory of amoeboid motion . Science the major processes in amoeboid locomotion, in- (Wash . D. C.) . 177 :637 . cluding the patterns of cytoplasmic streaming, the 5. HuxLEY, H . E. 1969. The mechanism of muscular contraction . Science (Wash . D . C.) . 164 :3561 . site of the generation of the motive force, the loca- 6 . ALLEN, R . D., J. W. COOLEDGE, and P . J . HALL . tions of steps in the rheological cycle, and the role 1960. Streaming in cytoplasm dissociated of the plasmalemma in locomotion and cell attach- from the giant amoeba, Chaos chaos . Nature ment. (Lond . ) . 187 :896 . Many unsolved problems remain . How pseudo- 7. SIMARD-DUQUESNE, N., AND P . COUILLARD . podium formation begins is still unknown as are 1962 . Amoeboid movement. I . Reactivation the factors that control the number and form of of glycerinated models of Amoeba Proteus with pseudopodia . Much remains to be learned about adenosine-tri phosphate . Exp. Cell Res. 28 :85 . the respectives roles of the cytoplasm and the 8 . SIMARD-DUQUESNE, N ., and P . COUILLARD . 1962. Amoeboid movement . II . Research of plasmalemma in excitability and behavior . It can . be seen that the extended frontal contraction contractile proteins in Amoeba proteus . Exp Cell Res. 28 :92 . theory offers some fruitful insights into mechanisms 9. THOMPSON, C. M ., AND L. WOLPERT . 1963. The of behavior (54) . isolation of motile cytoplasm from Amoeba At the molecular level, the thick filaments need Proteus. Exp. Cell Res. 32 :156 . to be characterized in order to establish their 10. POLLARD, T. D., and S . ITO. 1970 . Cytoplasmic identity by biochemical criteria . Similarly, the filaments of Amoeba Proteus . I . The role of macromolecular basis of calcium control remains filaments in consistency changes and move- obscure . It is not known whether amoeba cyto- ment . J. Cell Biol. 46 : 267 . plasm contains a system of calcium-sensitive pro- 11 . NACHMIAS, V. T . 1964 . Fibrillar structures in the teins similar to or analogous with the troponin- cytoplasm of Chaos chaos . J. Cell Biol. 23 :183. tropomyosin system of vertebrate striated muscle 12 . NACHMIAS, V . T. 1968 . Further electron micro- (55-57) or whether the light chains of myosin scope studies on fibrillar organization of the ground cytoplasm of Chaos chaos. J . Cell Biol. might be responsible for calcium control as in 38(l) :40. more primitive muscles (58) . Experiments are 13 . POLLARD, T . D., and E . D . KORN . 1971 . Fila- now in progress to find answers to these questions . ments of Amoeba proteus. II . Binding of heavy The authors are very grateful to Professors Philip B . meromyosin by thin filaments in motile cyto- Dunham, Charles Edwards, and Frederick G . Walz, plasmic extracts . J. Cell Biol. 48 :216 . Jr ., and to Dr . Suzanne M . Pemrick for advice 14 . COMLY, L . T. 1973 . Microfilaments in Chaos throughout the present study. We also wish to thank Carolinensis membrane association, distribution, Mrs . Alice C . Jacklet, Mrs . Marcia W . Cockrell, and heavy meromysin binding in the glyceri- Messrs . Dale T. Rice, Ryland W. Loos, Robert G. nated cell . J. Cell Biol. 58:230 . Speck, and Robert M. Zeh for technical assistance . 15 . GRIFFIN, J . L . 1964 . In Primitive Motile Systems This work was supported by research grant GM in Cell Biology. R. D . Allen and N . Kamiya, 18854 from the National Institute of General Med- editors . Academic Press, Inc ., New York . ical Science . 303 . Received for publication 30 March 1973, and in revised 16 . GICQUAUD, C. R ., and P. COUILLARD . 1970 . form 10 July 1973. Preservation des mouvements dans le cyto- plasme demembran6 d' Amoeba proteus . Cyto- REFERENCES biologie. 1 :460. 17. BARANY, M ., and K 1 . ALLEN, R. D . 1972 . In The Biology of Amoeba. . BARANY . 1970 . Change in J. Jeon, editor. Academic Press, Inc ., New the reactivity of myosin during muscle con- York . 201 . traction. J. Biol. Chem. 245 :2717. 2 . JAHN, T . L . 1964. In Primitive Motile Systems 18. MARSTON, S . B ., and R . T. TREGEAR . 1972 . in Cell Biology, R . D . Allen and N . Kamiya, Evidence for a complex between myosin and editors . Academic Press, Inc., New York . 279 . ADP. Nat . New Biol . 235 :23 . 3 . JOHN, T. L ., and J . J. VOTTA . 1971 . Capillary 19. WEBER, A ., and S . WINICUR . 1961 . The role of suction test of the pressure gradient theory of calcium in the superprecipitation of actomyo- amoeboid motion . Science Wash . D. C. 177:636 . sin . J. Biol. Chem . 236 :3198 . 4 . KIRBY, G . S ., R . A . RINALDI, and I . C. CAMERON . 20 . WEBER, A ., and R . HERZ . 1963 . The binding of 1971 . Capillary suction test of the pressure calcium to actomyosin systems in relation to

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