Pattern of birefringence in the giant amoeba, Chaos carolinensis

Experimental Cell Research 72 (1972) 34-45

PATTERN

OF BIREFRINGENCE CHAOS

of Biological

Sciences,

State

AMOEBA,

CAROLINENSIS

ROBERT Department

IN THE GIANT

University

D. ALLEN

of New

York

at Albany,

Albany,

N.Y.

12203,

USA

SUMMARY 1. The giant amoeba, Chaos carolinensis, shows a dynamic but characteristic pattern of weak birefringence demonstrable in polarized light photomicrographs only after light scattering inclusions have been removed. 2. The plasmalemma, presumably because of its ‘fringe’ layer, has slightly stronger birefringence than the membranes of vesicles. The slow axis of transmission is parallel to the membrane surface. 3. The streaming endoplasm always shows positive axial birefringence (slow axis parallel to the stream) along its entire length. When streaming stops, this birefringence decreases. 4. The ectoplasmic tube has weaker birefringence and its sign varies locally. The peripheral region of the ectoplasmic tube is usually positively birefringent. Negative birefringence, if observed, has a characteristic patchy distribution localized mostly adjacent to the endoplasmic stream. 5. Formation of a new pseudopod results in the rapid establishment of the pattern of birefringence described above. When the pseudopod begins to retract, the entire pattern fades until it disappears in the light-scattering noise of remaining cytoplasmic mclusions. 6. The pattern of birefringence is the only reliable evidence now available on the deployment and orientation of submicroscopic linear elements in the cell and their behavior during amoeboid movement. The results are entirely consistent with the frontal contraction theory of amoeboid movement.

As a Postdoctoral Fellow of the US Public Health Service in Professor John Runnstriim’s laboratory in 1954, I made the fortuitous discovery that the cytoplasm of a giant amoeba could stream vigorously when freed from the confines of its own cell membrane and enclosed in a glass capillary [8]. When I demonstrated this to Professor Runnstrom, he shared my excitement and made two comments that shaped the direction of my work for many years. First, he said that it was often just such unexpected observations that led eventually to the solution of fundamental biological problems. Then he suggested that I examine amoeba cytoplasm in polarized light for the possible presence of birefringent fibrils that might constitute part of a contracExptl

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Res 72

tile system. Professor Runnstrijm later gave me instruction in the theory and use of the polarizing microscope, an instrument with which he himself had done pioneering work many years before. The work reported here is thus a direct outgrowth of work begun in the laboratory of Professor Runnstrbm, whose memory is honored by this volume. Until recently, the sensitivity of polarizing microscopes has been insufficient to detect the pattern of birefringence in most amoeboid cells. Mitchison [22], for example, found only membrane birefringence in Amoeba proteus. Since that time the factors influencing the dynamic range of polarizing microscopes have been identified by Inod [18], with the result that with careful selection from among im-

Birefringence in Chaos carolinensis proved commercially available components, it is possible to assemble a highly sensitive polarizing microscope capable of recording phase retardations well below 1.0 A. The fact that amoebae move rapidly has made it impossible to obtain high quality photographic records of their pattern of birefringence using conventional light sources. However, the recent introduction of the phase randomized laser light source [20] has shortened the required photographic exposure time by between one and two orders of magnitude. The pattern of birefringence due to the alignment of linear elements of the contractile system in the giant amoeba is a classic example of a weak ‘signal’ buried in ‘noise’, due in this case to inclusions which both scatter and depolarize light. One way to recover the signal is to discard the noise electronically by the application of synchronous detection techniques to a microbeam measurement method [7]. This method, phase modulating microphotometry, has been applied, for example, to the study of strain birefringence in the viscoelastic endoplasm [14]. It is currently being applied to the measurement of birefringence changes due to dynamic behavioral events in the cell (e.g., reversal, stimulation, etc.). Another way to recover the signal is to reject the noise biologically by micrurgical removal of a substantial portion of the light-scattering inclusions of selected cells [4]. While both approaches are valuable, the latter has the advantage that polarized light images readily display the geometrical pattern of birefringence in a large portion of the cell.

MATERIAL

AND

METHODS

Chaos carolinensis were raised in mass cultures using Marshall’s medium [4]. Tetrahymena were added as food every 48 h. Amoebae were starved for a day before experiments, then centrifuged at about 550 g

35

in a conical centrifuge tube with a cushion of 1 4, agar on the bottom for 1 min. They werethen pipetted onto a cleanslide and each wasquickly bisectedwith a glassknife in such a way that a ‘clear’ fragment containing especiallysome nuclei and mitochondria was separated from the remainder of the cell, which was densely packed with heavy inclusions, suchas triuret crystals 1161 and refractile bodies [ll, 121. The method was describedmore fully by Allen & Francis t41.

A ZeissphotomicroscopeI was usedwith a selected Abbe-type ‘pol’ condenseroiled to the slide and the following achromatic strain-free objectives:6 x (N.A.

0.16), 10x

(N.A. 0.25), 16 x (N.A. 0.33) and 4Oi~

(0.85 oil) For the three lower powers, the systemwas cleaned and adjusted until the extinction factor exceeded 10’. Because amoebae move rapidly, and because two photographs at two opposite compensator settings are required to demonstrate birefringence, the conventional mercury source was replaced by the 514 nm line of a 5 watt CW argon-ion laser, Spectra-Physics model 265. A beam of light from a laser is too coherent to form a recognizable microscopic image. Consequently, the beam was phase-randomized by passing it through a spinning roughened plastic disc [20]. The resulting partially coherent illumination provided images of at least comparable quality to those obtained with a high pressure mercury arc line source and had a brightness up to 100 times greater. Required photographic exposures were of the order of a second on plus-X Kodak 35 mm negative film processed in diafine. The images recorded in figs 1-6 are useful only for semi-quantitative estimates of birefringence in different regions of the cell. Efforts to make quantitative measurements from the films by the method of Allen & Nakajima [5] were defeated by the fact that too much time was required to measure precisely the extinction angle as well as the bias compensations at which the pairs of photographs were taken. This was because the very weak birefringence of the slide and coverglass caused the background extinction angle to vary as the specimens changed their direction of movement. Because of these factors, the compensator was set by eye, and considerabletraining and practice were required to find repeatably the bias compensation settings for maximum contrast [7]. When these were recorded electronically by a potentiometer attached to the dial of the n/30 Brace-Kiihler compensator, it was found that the bias settings varied between 1.5 and 3.0 degrees.

RESULTS Specimens selected at random from mass cultures for examination in polarized light do not show conspicuous signs of birefringence except in their plasmalemmas. Food vacuoles, crystals and refractile bodies scatter so much light that even contrast due to the membrane Exptl

Cell Res 72

36 R. D. Allen

1. Composite photomicrographs at opposite compensator settings of a long lobopodium of Chaos curolinenshowing its weak positive axial birefringence. P, plasmalemma; HE, hyaline ectoplasm; N, nucleus; GE, granular ectoplasm; ES, endoplasmic stream; DF, dorso-lateral fin. Change in contrast at the junctions of composite photographs are due to small variations in bias retardation over the field at low power. Inserts show the orientation, with respect to the specimen, of the polarizer(P), analyzer (AN) and the slow axis of thecompensator (S).

Fig. sis

may be swamped. Both starvation and micrurgical removal of a substantial portion of these inclusions are required in order to observe birefringence in the ground cytoplasm. Even Exptl

Cell Res 72

these treatments produce variable results. Consequently, a selection must be made of from two to five cells from among 25-30 prepared for observation. This selection is

Birefringence

in Chaos carolinensis

37

Fig. 2. Three views of a large Chaos with a compound leading pseudopodium. A and a show the tail region, Band b the middle, and C and c show the advancing pseudopodium. MF, membrane folds; CR, contrast reversal due to orthogonal orientations; HC, hyaline cap; S-, streaming direction.

not for the most highly birefringent cells, but for those with the combination of low light scatter and high motile vigor. Under low power objectives (6 x, 10 x, 16 > ), Chaos pseudopodia show as a whole weak, positive, axial birefringence which is more obvious in the plasmalemma than in either the granular ectoplasm or endoplasmic stream. The contrast visible in the hyaline

ectoplasmic region is probably due in large part to the over-and underlying plasmalemma (fig. 1). A feature of pseudopod shape that presented problems in this study was the membrane folds that may extend along the dorsal or lateral margins of the pseudopod posteriorly from a point within a few hundred microns from the tip. Birefringence due to Exptl

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R. D. Allen

Fig. 3. The base of a thick pseudopodium with a single endoplasmic shows weaker birefringence than the plasmalemma (P).

these membrane folds may appear as spurious diffuse cytoplasmic birefringence at optical section levels well within the cell interior. Consequently, the presence or absence of these folds had to be determined by focusing through the specimen before photographs were taken, and their presence noted. In fig. 2, it can be seen that the slow axis of transmission is parallel to the plasmalemma and other cell membrane surfaces and to the pseudopod axis in the endoplasmic stream, and in the peripheral portion of the ectoplasmic tube. The positive axial birefringence of the streaming endoplasm is easily seen in fig. 2, B and b, where contrast reversal (CR) occurs at orthogonal orientations. Note that Exptl Cell Res 72

stream (ES). A vesicle membrane (V)

even small non-motile pseudopodia are birefringent. Streaming endoplasm is birefringent all the way from the advancing pseudopod tip to the tail where the endoplasm is recruited from the walls of the ectoplasmic tube. This is detectable with 6 x and 10 x objectives, figs 1 and 2, but is more easily seen with a 16 x objective. In fig. 3, for example, endoplasmic birefringence is unmistakable. In the same figure it can be seen that the birefringence of the ectoplasmic tube is less strong, and of variable sign. By comparing the brightness of the ectoplasmic region to background in the paired photographs, it can be seen that there are patches of birefringence of both signs.

Birefringence in Chaos carolinensis

39

Fig. 4. A thick pseudopodium with one endoplasmic stream (ES) and membrane folds (MF). Note the positive axial birefringence in the endoolasmic stream and the negative axial birefringence in the adjacent region (X) of the ectoplasn& tube.

This is shown more clearly jn fig. 4, where the region “x” immediately adjacent to the endoplasmic stream is negatively birefringent, i.e. has its fast axis parallel to the pseudopod axis, while a more peripheral region is positively birefringent. This situation appears to be typical. Fig. 5 shows the changes in the birefringence of newly forming, advancing, and retracting pseudopodia. Paired frames 5A and 5a show the positive birefringence of the endoplasm along the main body. During the exposure of 5a, an endoplasmic stream flowed for a short time from the base of the right pseudopod (R) into the middle (M) and left pseudopod (L). This stream showed strong birefringence for a period of less than 2 sec.

The specimen was rotated just before frames 5 B and b. The new orientation places emphasis on the birefringence of pseudopods R and L, which are advancing and retracting respectively. Birefringence in the main body and middle pseudopod M produce hardly any contrast at this orientation where the crystalline axes are parallel to the polarizer. The positive birefringence of extending right pseudopod R continues to be strong except for patches of reversed sign which are almost certainly ectoplasmic. The birefringence of the retracting left pseudopod L is weaker than R at all times and diminishes with time. An unusual feature of this cell was its unusually large isotropic hyaline cap region. The patchiness of the birefringence in the Exptl Cell Res 72

40

R. D. Allen

F;g. 5. A sequence of four pairs of photographs taken at opposite compensator settings and showing the development of relatively strong birefringence during one case of pseudopod extension and retraction. Note in frames A and n, the strong positive axial endoplasmic birefringence visible almost into the tail, and the weaker negative axial birefringence adjacent to the stream. A pattern of positive birefringence is visible in the cytoplasm streaming into the left (L) and middle (M) pseudopodia. In frames B and b, the specimen has been rotated to emphasize the changing pattern of birefringence in advancing pseudopod (R) and one that is retracting (L). In the advancing pseudopod note the strong birefringence throughout, except the unusually wide isotropic hyaline cap zone at the tip. In the retracting pseudopod (L), note the gradual diminution of positive birefringence.

granular ectoplasm is especially well shown along the body in 54 where the ectoplasm adjacent to the endoplasm is negatively birefringent in contrast to that found more peripherally. Fig. 6B shows that both the patchiness and strong positive axial endoplasmic birefringence extend well into the tail region. There is one form of patchiness which occurs prominently in amoebae showing sporadic streaming. It is not uncommon to find lateral strips or lines of negative axial bireExptl Cell Res 72

fringence across the anterior rim of the ectoplasmic tube (fig. 7). The strips generally move backward a few microns from the point at which they are first detected, then sometimes fade or disappear. It appears from all live observations and all photographs examined that the plasmalemma is of more or less uniform birefringence over the entire amoeba. Variations in contrast in the figures are believed to be due to geometry of the cell and small variations

Birefringence in Chaos carolinensis

41

Fig. 6. Advancing pseudopod (A, a) and tail region of a small specimen with unusually strong positive axial endoplasmic birefringence extending from the frontal region to the recruitment zone of the tail. Note also patches of negative birefringence in the ectoplasm.

in the bias retardation over the microscopic field at low power (see especially fig. 1). There is, however, a clear difference between the birefringence of the plasmalemma and that of vesicular membranes (fig. 3), the former being more birefringent, presumably due to the presence of the ‘fringe’ layer.

DISCUSSION The rationale for the examination of amoeba cytoplasm in polarized light has been to look for a pattern of birefringence as the most reliable indication of the orientation and concentration of linear (fibrillar) elements in the Exptl Cell Res 72

42

R. D. Allen

Fig. 7. An advancing pseudopod in which sporadicstreaming had establisheda seriesof birefringent linesalong the advancing rim of the ectoplasmictube. They are negativelybirefringent with respectto the pseudopod axis. Note also the apparent lines of positivelybirefringent strain in the frontal endoplasm.

living amoeba. A number of studies on fixed Chaos and Amoeba with the electron microscope [17, 23, 24, 25, 271 have shown the presence of 50-70 8, microfilaments and 140 A thick filaments. The thin filaments of A. proteus have been shown to bind heavy meromyosin from muscle and therefore are presumably actin [26]. However, these giant Exptl Cell Res 72

amoebae are notoriously resistant to conventional fixation. During the 20-30 set required for streaming to stop, the cytoplasm contracts locally, producing large abnormal hyaline blebs beneath the plasmalemma. L. Comly, in our laboratory, has shown that cytoplasmic birefringence is destroyed during fixation. Consequently, we can rely on the

Birejringence in Chaos carolinensis

electron microscope to tell us what structures may be present but not how the various elements of the contractile system are organized in the living state. The birefringence generally found in cells is largely a mixture of intrinsic and form birefringence [13, 281. Intrinsic (or crystalline) birefringence is that which remains when fixed doubly refracting material is immersed in a penetrating fluid of matching refractive index. The form birefringence component of a structure is that which varies when the refractive index of the immersing fluid is changed. From electron microscopic studies on fixed cells we assume that partially oriented thick and thin filaments in living Chaos cytoplasm produce a mixture of intrinsic and form birefringence. So far, however, this assumption has not been confirmed because fixation itself destroys virtually all cytoplasmic birefringence in the amoeba. The fact that the strongest birefringence found in the cell is found in the endoplasm when it is streaming suggests that some other form of birefringence may be involved. Since flow birefringence can be induced by velocity gradients in fluids containing asymmetrical particles, it might be natural to assume that the birefringence observed in streaming endoplasm is flow birefringence. Alternatively, the birefringence might be due to strain by virtue of the preferential alignment of cross-linked linear elements parallel to applied tension. A direct test of these alternative possibilities has recently been carried out by the measurement of endoplasmic birefringence while the endoplasm was caused to flow by the application of sharp suction to pseudopod tips [14]. The result was decisive in showing that birefringence increased not in proportion to the induced velocity gradient as expected according to the flow birefringence hypothesis, but in proportion to the duration of suction as would be expected of viscoelastic fluid under

43

stress. Release from suction caused elastic recoil of the endoplasm and a gradual drop in its birefringence. These findings demonstrate that the endoplasmic stream is viscoelastic and thus capable both of transmitting and storing tensile forces. The application of tension doubtless causes the temporary reorientation of linear elements so that more of them lie parallel to the axis of tension. In molecular terms, viscoelasticity indicates that extensive cross-linkage must occur between linear elements. There are other reasons for ruling out flow birefringence. First, the velocity gradients found in Chaos endoplasm do not exceed about five reciprocal seconds [6], a value one or two orders of magnitude less than the minimum shear gradients used in studies of macromolecular asymmetry. Second, when streaming stops suddenly, the birefringence takes several seconds to decay [ 141. Third, the entire axial endoplasm is birefringent, not merely the peripheral portion where the highest velocity gradients are found [6]. The pattern of birefringence found in this study is entirely consistent with the frontal contraction model of pseudopod formation and amoeboid movement [l]. According to the model, the motive force for cytoplasmic streaming and pseudopod extension is delivered as a contraction of cytoplasm approaching each pseudopod tip. As the viscoelastic endoplasm advances toward the pseudopod tip, it begins simultaneously to contract and undergo syneresis [3,4] while still in the axis of the pseudopod but finishes these processes soon after becoming everted to form the extending ectoplasmic tube. This contraction is thus so situated that it applies tension to the endoplasm and compression to the advancing rim of the ectoplasmic tube. Direct evidence for the existence of these forces was obtained from an earlier polarized light investigation [9], in which a cycle of detectable Exptl

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R. D. Allen

birefringence changes was found to accompany sporadic but not continuous streaming at pseudopod tips. Under those circumstances, positive birefringence appeared in the endoplasm and patches of negative birefringence in the ectoplasmic tube in just the manner expected if the postulated forces were acting to cause photoelastic effects (strain hirefringence) in a nearly isotropic cytoplasmic gel. The pattern of birefringence in a typical pseudopod is as follows. The endoplasm is relatively strongly birefringent (ca 10-j). When the endoplasm becomes everted to form the ectoplasmic tube, the latter has either a greatly reduced positive axial birefringence, or it may have local regions of changed sign. Often the birefringence of the ectoplasmic tube is patchy, with weak positive axial birefringence occurring near the periphery and weak negative axial birefringence adjacent to the endoplasmic stream. It is likely that this pattern results in part from the geometry of endoplasmic eversion: the axial endoplasm moves the longest distance to the periphery of the ectoplasmic tube and so suffers the least compression, while the peripheral endoplasm travels the shortest distance and must therefore undergo the greatest degree of compression. Why is the ectoplasmic birefringence patchy? The patches of negative axial ectoplasmic birefringence have been observed to form as the ectoplasm is laid down at the front of a pseudopod [9]. Presumably they are due to compression of a portion of the cytoplasmic gel such that the linear elements in the gel assume a mean orientation that is no longer parallel to the pseudopod axis but orthogonal to it. To account for a radially asymmetrical pattern of birefringence, it is necessary to assume that the force is not applied with radial symmetry. The details of pseudopod extension bear this out, for neither Exptl

Cell Res 72

the thickness of the ectoplasmic tube nor the volume of lateral flow of cytoplasm at the tip is radially symmetrical. In films of pseudopod extension, the endoplasm seems to shift laterally near the tip. It has even been observed in a few exceptional cases that pseudopodia may form in helical rather than radial symmetry. We have not been fortunate enough to observe such a pseudopod in polarized light, but a helical band of ectoplasmic negative birefringence might be expected in such a cell. The finding that endoplasmic birefringence extends into the recruitment zone of the amoeba’s tail appears to verify the earlier assumption [I] that the linear elements in the entire endoplasm may be oriented due to the transmission of forces applied at pseudopod tips. Before the recent demonstration that strain birefringence could be induced in the endoplasm [14], this facet of the frontal contraction model was less than totally convincing. There would appear to be only three ways that the contractile system known to be present in giant amoebae could bring about cytoplasmic streaming into pseudopodia: a pressure gradient, active shearing along the walls of the ectoplasmic tube, and contraction of the everting endoplasm at pseudopod tips. The pressure gradient theory [15, 19, 211 has been invalidated by recent experiments showing that reversal of the assumed pressure gradient by suction does not cause cytoplasmic streaming to reverse [lo]. The pressure gradient theory, which gained popularity despite its failure to account for several aspects of amoeba behavior, has thus received an experimental coup de g&e. The active shearing model, first considered as an unlikely possibility by Allen [l] has recently been proposed seriously by Subirana [29], who developed his model so far as to predict the kind of velocity profile to be expected in

Birefringence in Chaos carolinensis

amoebae. Unfortunately, this author was not aware that the velocity profiles for cytoplasmic streaming in amoebae had been determined previously [6] to be of a type directly in conflict with that demanded by the model. Therefore, there seems to be no compelling reason to reconsider the active shearing model in the case of amoeboid movement. What is left among contractility models, then, is the frontal contraction model, which was originally proposed because it was consistent with the known behavior of amoeboid cells [I, 21 and of cytoplasm isolated from the cell [8]. The frontal contraction model has now been subjected to a number of experimental tests and has survived these sufficiently well to consider elevating it to the status of a theory which has been shown to be substantially correct. I would like to thank Dr Hiromichi Nakajima and Dr David Francis, who assisted in the very early phases of this work at Princeton University. I should also like to thank Professor Shinya Inoue and Professor Gordon Ellis for their suggestions. This work was made possible by Research Grant GM-08681 and by Program Grant GM-14891 from the National Institute of General Medical Science.

REFERENCES 1. Allen, R D, Exptl cell res, suppl. 8 (1961) 17. 2. - Sot exptl biol symp 22 (1968) 15 I.

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3. Allen, R D & Cowden, R R, J cell biol 12 (1962) 185. 4. Allen, R D & Francis, D W, Sot exptl biol symp 19 (1965) 259. 5. Allen, R D & Nakajima, H, Exptl cell res 37 (1965) 230. 6. Allen; R D & Roslansky, J D, J biophys biochem cytol 6 (1959) 437. 7. Allen, R D, Brault, J W & Zeh, R. Outical and electron microscopy (ed R Barer & V’Cosslett). Academic Press, New York (1966). 8. Allen, R D, Cooledge, J W & Hall, P J, Nature 187 (I 960) 896. 9. Allen, R D, Francis, D W & Nakajima, H, Proc natl acad sci US 54 (19651 1153. 10. Allen, R D, Francis,‘D W & Zeh, R, Science 174 (1971) 1237. 11. Andresen, N, Compt rend trav lab Carlsberg 24 (1942) 139. 12. - Ibid 29 (1956) 435. 13. Bennett, H S, McClung’s handbook of microscope technique, 3rd edn. Hoeber, New York (1950). 14. Francis, D W & Allen, R D, J mechanochem cell motility 1 (1971) 1. 15. Goldacre, R J & Larch, I J, Nature 166 (1950) 497. 16. Griffin, J L, J biophys biochem cytol 7 (1960) 227. 17. - J cell biol 27 (1965) 39A. 18. Inoue, S, The encyclopedia of microscopy (ed G L Clark). Reinhold, New York (1961). 19. Jahn, T L, Primitive motile systems’in cell biol (ed R D Allen & N Kamiva) u. 279. Academic Press, New York (1964). _ . 20. LaFountain. J, Muckenthaler. F & Allen. I R D. -> J cell biol 8’(1968) 159A. 21. Mast, S 0, J morph01 physiol41 (1926) 347. 22. Mitchison, J M, Nature 166 (1950) 313. 23. Nachmias, V T, J cell biol 23 (1964) 183. 24. - Ibid 38 (1968) 40. 25. Pollard, T D & Ito, S, J cell bio146 (1970) 267. 26. Pollard, T D & Korn, E D, Ibid 48 (1971) 216. 27. Schafer-Daniel, S, Z Zellforsch 78 (1967) 441. 28. Schmidt, W J, Die Doppelbrechung von Karyoplasma, Zytoplasma und Metaplasma. Protoplasma-monographieen, vol. 11. Berlin (1937). 29. Subirana, J A, J theor biol 28 (1970) 111.

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