Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli

466 Short notes

Exp Cell Res 147(1983)

SHORT NOTE Quantitative Analysis of Changes in Cell Shape of Amoeba proteus during Locomotion and upon Responsesto Salt Stimuli

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form resewed 0014-4827183 $03.0010

TETSUO UEDA and YONOSUKE KOBATAKE Department of Pharmaceutical Sciences, Hokkaido University. Sapporo 060, Japan A new parameter expressing the complexity of cell shape defined as (periphery)‘/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5x 10m3Hz. The complexity increased mainly due to elongation of the cell as external Ca2+ increased. This effect was blocked by La3’, half the inhibition being attained at l/200 amount of the coexisting Ca‘+. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as S?+, Mg*+, Co*‘, Ni2+, Naf, K+ etc., increased. Irrespective of the opposite effects of Ca*+ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.

Amoeba proteus and other amoeboid cells change their cell shape during locomotion and in response to external stimuli. So far, this has been described qualitatively using terms such as ‘branching’ in starved conditions, ‘star state’ in phagocytosing food organisms, or ‘rounding up’ in adverse environments [l-3]. The available quantitative description of amoeboid motility is based on a random walk model as applied to Amoeba proteus [4], fibroblasts [5], granulocytes [6], etc. However, this method can only be applied to a long-term locomotion, and pays no regard to changes in cell shape, a specific character in amoeboid motility. In this paper we show that the complexity of cell shape, a non-dimensional quantity defined as (periphery)2/(area) in 2D projection, is a useful parameter for characterizing changes in cell shape of Amoeba proteus quantitatively during both short- and long-term locomotion and upon responses to chemical stimuli. Materials and Methods Organisms. Amoeba proteus was kept in a basal solution containing 10m4M KCl, CaC12 and MgS04 at pH 7.0, and fed on Tetrahymena [7]. The organisms were washed with the basal solution 2-3 times before use. Determination of the complexity of cell shape of Amoeba. The organism was placed at the center of a shallow glass trough (30x30 mm*) which contained the basal solution or a solution to be tested, viewed under an inverted microscope with a magnification 20x7, and photographed. The periphery of the cell in the picture was traced with about two hundred points on a digitizing pad (Houston Instrument, model, HiPAD-02Y-O182), and the data were stored and analysed with a microcomputer (Nippon Electric Co., model PC-8801). The value of the complexity was calculated according to the formula; Complexity=(periphery)*/(area). Preliminarily we tried some other parameters, such as moment of inertia, area or periphery for describing changes in cell shape quantitatively. All these parameters depended on the size of a cell, giving different values even for similar cell shape when the dimension is different. Thus, averaging and accumulation procedures over many organisms were meaningless in these cases. Therefore, introduction of a dimensionless quantity was critical. The complexity defined above is the simplest parameter having both the required character and clear physical meaning.

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Exp Cell Res 147 (1983)

Measurements of the motive force of the protoplasmic streaming in amoeba. The motive force of protoplasmic streaming in amoeba was measured by the double chamber method proposed by Tasaki & Kamiya [8]. An amoeba was sucked at the middle of the opening of a tire-polished glass capillary (about 30 urn 0). A difference in hydrostatic pressure was applied to just counterbalance the protoplasmic streaming, and recorded on a penwriting recorder through a pressure transducer (Statham Instruments Inc., USA, Model SClOOl, Model UPG 4). The Fourier analysis of changes in the complexity and the motive force. According to the theory of the Fourier transformation, any quantity g(t) varying with time, 1, can be expanded by sin and cos functions with different frequency f: oc g(r)

= I

cct) =

-I

WI exp(i2n fr) dJ g(t) exp( -i2n ft) dt,

(1) (2)

and i is a unit imaginary number. The power spectrum density function P(j) is defined as squared absolute value of the Fourier transform Gy) per unit time: (3) and expresses a fraction of a component oscillation having a frequency f. Experimentally we get a finite number of experimental points N, and in this case eqs (I), (2) and (3) are rewritten as g(nlN) = 2 g( k/N) exp( i2xnklN) t=o = a, + 2 { a(klN) cos (2wzklN) + ib( k/N) sin (2JmklN)) k=O

G(n/N) = l/N $g(k)

exp(-i2xnklN)

k=O

P(nIN) = IIN }G(nlN)(?

16)

The power spectrum was obtained by use of an g-bit microcomputer program for the Fast Fourier Transformation. Determination of intracellular ATP concentration. About 2 000 amoebae suspended in 0.5 ml of the respective test solutions were heat-killed by adding an equal amount of hot buffer (10 mM Tris-HCl, pH 7.0), and boiled for 10 min to extract ATP. The suspension was centrifuged (1500 g) for 10 min, and the ATP concentration in the supematant was measured photometrically by the luciferin-luciferase method [9]. All physiological experiments were performed at 21f1°C, repeated more than four times at each experimental condition.

Results Periodic changes in cell shape of Amoeba proteus during locomotion. A series of pictures taken at 20-set intervals during locomotion of Amoeba proteus are shown in fig. 1A. Two phases of changes in cell shape appear alternately; in one phase (a-c; i-k) the cell elongates, is less branched and consequently migrates fast, and in the other (d-h) the cell is rather round and branched and thus can change the direction of locomotion. Corresponding to this, the complexity of cell shape changes periodically, as shown in fig. 1B. Here, oscillation with a period of about 5 min dominates, and

468 Short notes

Exp Cell Res 147(1983)

every

Time (min)

A

Amoeba

s B

R y Physarum

20 set

Fig. I. Changes in cell shape of A. proteus during locomotion. (A) A series of micrographs taken at 20set intervals. The first picture is at 3.0 min in (B). (B) A time course of the complexity of the cell shape. The complexity is defined as (periphery)‘/(area) in 2D projection. Fig. 2. Time courses of (A) the motive force of the protoplasmic streaming in A. proteus; (B) of the tension generation in a plasmodial strand of Physarum. The latter was measured as previously reported

1181.

the Fourier analysis demonstrates this point quantitatively, as shown in fig. 3A. This period is similar to that determined from a random walk model [4]. As clearly seen in figs 1B and 3 A, regular oscillation does not persist for a long time. Thus, changes in cell shape of Amoeba proteus have a short-range, but not a long-range regularity. Periodic changes in the motive force of protoplasmic streaming of Amoeba. Time courses of the motive force of protoplasmic streaming in Amoeba proteus and of the tension generation in Physarum plasmodium are shown in fig. 2A, B, respectively. The oscillation in Amoeba is not so regular as that in Physarum, but does exhibit a certain degree of regularity. The power spectra of these oscillations in Amoeba and Physarum are shown in fig. 3 B, C. There is a sharp single peak in Physarum, while there is a broad peak in Amoeba. As shown in the inset table, in frequency distribution SD amounts to about 50% in Amoeba, while that in Physarum is about 10%. The pattern of the power spectra for changes in cell shape during locomotion and the motive force in Amoeba resemble one another, as seen in fig. 3A, B, and this indicates that the contractile apparatus of Amoeba exhibits a certain degree of rhythmicity. Roles of Ca2+ and ATP in the regulation of cell shape in Amoeba. External

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(1983)

T 2.4 2.9 7.1

in Amoeba a b c -

motive

0

2

4

unit

SD 1.2 1.3 074 lo-3Hz

Fig. 3. Spectral analyses of the amoeboid motility. (A) Complexity in Amoeba (fig. 1E); (B) motive force of the protoplasmic streaming in Amoeba (fig. 2A); (C) tension generation in the plasmodial strand of Physarum (fig. 28). Inset: List of mean and SD values.

force

6 Frequency

80

2

4

6

810

(10s3Hz)

salts affect the cell shape of Amoeba proteus, and in fig. 4 the complexity of cell shape is plotted against the logarithm of concentration for various cations. The insets are optical micrographs of amoebae immersed in respective salt solutions. The complexity increases gradually due to elongation and branching of the cell by Ca*+, but decreases or is not affected by all other cations examined, such as Sr*+, Mg*+, Co*+, Ni*+ Ba*+, Naf, Kf, Lif etc. Thus, the increase in the complexity may be a specific action of Ca*+, because even a chemically similar Sr*+ cannot be substituted. The elongation and branching of amoeba in high Ca*’ should be maintained by formation of cortical actin meshworks, and intracellular ATP, Ca*+ and/or pH may modify the structure. Among them correlation between intracellular ATP concentration and cell morphology was tested, and the results obtained are shown in fig. 5. Divalent cations affect the ATP level at ten times lower concentrations than monovalent cations, and irrespective of the opposite action on the cell shape (compare figs 4, 5), the relative ATP concentration decreased for both Ca*+ and other cations. Therefore we conclude that there is no correlation between cell morphology and net ATP concentration. The presence of La3+, a calcium channel blocker, inhibited elongation of the cell by Ca*+ . The dose-response curve of this inhibition is shown in fig. 6, where the complexity of cell shape 30 min after the treatment is plotted against the La3+ concentration at various levels of Ca*+ concentration. The higher the Ca*+ concentration, the higher the quantity of La3+ required to inhibit the Ca*+ effect. This inhibitory power of La3’ is now defined as the concentration of La3+ to reduce half the complexity at a given Ca*+. Double logarithmic plots between the Ca*+ concentration present and the inhibitory power of La’+ give a linear relationship with the unit slope, as shown in fig. 6, inset. From this result, about l/200 less La3+ than the coexisting Ca*+ is shown to block the Ca*’ action on the cell morphology. Our results are consistent with the observation by Nuccitelli et al. [lo] that Ca*’ passes into the cell at the tail region, and suggest that Ca*+ thus taken up

Exp Cell Res 147 (1983)

470 Short notes

OL

’ -4

-3

-2

,L.--.-L -4

-1

-3

-2

Ca

-1

log (concentration.M)

log koncentration.M) P

6 !? w-u-t

G-2

log (LaCl3 concn. MI Fig. 4. Dependence of the cell shape of A. proteus on external salts. The complexity of the cell shape

is plotted against log concentration of stimulus chemicals which were dissolved in the basal solution. Data for Co*+, Ni*+, Ba*+ and Li+ fall within the region between Na and Sr, Mg (not shown). Data at each concentration are the averages over about 15 amoebae, and the SD are about 10%. Fig. 5. Intracellular ATP concentration as a function of log concentration of stimulus chemicals. Experimental conditions are the same as in fig. 4. Fig. 6. Effects of La3+- on Cazi-induced changes in the cell shape of A. proteus. The complexity of the cell shape is plotted against log La3+ in the presence of various Ca2+ concentrations. Inset: Double logarithmic plots between Cazc and the inhibitory power of La’+, showing a line with a unit slope.

acts on the actomyosin system to form the tail morphology of the cell. Actually the elongated and branched amoebae in high Ca*+ did not adhere to the glass substrate, except at the tip portion. This property resembles that of the tail portion in the normal morphology. Discussion By introducing the quantitative method for analysing changes in cell shape, we showed above that Amoeba oscillates during locomotion. This rhythmicity in Amoeba is an indication of synchrony or interdependence of the pseudopodia [ 111.Other amoeboid cells, such as leukocytes in higher animals, Acanthamoeba and the myxomycete Physurum plasmodia also locomotes with rhythmic contraction [ 12, 133. Thus, periodicity in amoeboid motility seems ubiquitous. Our quantitative method will be applicable to all amoeboid cells wherever there is a change in cell shape during locomotion, and will reveal dynamic aspects of this

Exp Cell Res 147 (1983)

Short notes

self-sustained oscillation of the contractile apparatus as a dissipative structure in the non-equilibrium system [ 141. The rhythmicity in Amoeba is less regular than in Physarum (see fig. 3). Physical conditions which lead to this irregularity might be either chaos occurred when a non-linear system is subjected to a condition far from the equilibrium [ 151 or interaction among oscillators within a cell [16]. In either case further study is needed. Our method will be improved technically by introducing a computer-linked video system. We are indebted to the late Professor F. Takeda at Hosei University and Dr H. Hayashi at Kyushu University for affording the organism and the FFT program, respectively. Thanks are also due to Mr K. Kikuchi for performing part of the experiments. This study was partially supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References 1. McLellan, M R & Morris, G J, Cryo-letters 3 (1982) 35. 2. Zimmerman, A M, Ann NY acad sci 78 (1959) 631. 3. Bovee, E C & Jahn, T L, The biology of amoeba (ed K W Jean) p. 249. Academic Press, New York (1973). 4. Sayers, Z, Robers, A M & Bannister, L H, Acta protozool 18 (1979) 313. 5. Gail, M H & Boone, C W, Biophys j 10 (1970) 980. 6. Peterson, S C & Noble, P B, Biophys j 12 (1972) 1048. 7. Prescott, D M & James, T W, Exp cell res 8 (1955) 256. 8. Tasaki, I & Kamiya, N, J cell camp physiol 63 (1964) 365. 9. Beutler, E & Baluda, M, Blood 23 (1%4) 688. 10. Nuccitelli, R, Poo, M M & Jaffe, L, J gen physiol 69 (1977) 743. 11. Grebecki, A & Klopocka, W, J cell sci 50 (1981) 245. 12. Senda, N, Tamura, H, Shibata, N, Yoshitake, J, Kondo, K & Tanaka, K, Exp cell res 91 (1975) 393. 13. Kamiya, N, Protoplasmatologia 8 (1959) 1. 14. Nicolis, G & Prigogine, I, Self-organisation in non-equilibrium systems. John Wiley & Sons, New York (1977). 15. Haken, H (ed), Chaos and order in nature. Springer-Verlag, Berlin (1981). 16. Tomita, K & Kai, T, J stat phys 21 (1979) 65. Received February 21, 1983 Revised version received May 30, 1983

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