Variations in myoneme birefringence in relation to length changes in Stentor coeruleus

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Experimental Cell Research 85 (1974) 127-135

IATIONS IN MYONEME BIREFRINGENCE LENGTH CHANGES IN STENT ~~~~~~~~~ BERIT I. KRISTENSEN,

LIS ENGDAHL

NIELSEN

and J. RQSTGAARD

Zoophysiological laboratories B and C, August Krogh Institute, and Anatomy Department C, University qf Copenhagen, DK 2100 Copenhagen 0, Denmark

SUMMARY The stalk segment of the heterotrich ciliate, Stentor coeruleus, appears nearly isotropic in the contracted state and develops a characteristic birefringence during extension. The birefringence occurs in stripes and is associated with the myonemes, one of the two longitudinally running subpellicular fiber systems. Electron microscopical investigations reveal changes in the ultrastructure of the myonemes from the extended to the contracted state. The relaxed myonemes consist mainly of 3 nm filaments running in the longitudinal direction, while the contracted myonemes show 10 nm tubular-like filaments, more randomly oriented. It is suggested that during elongation tbe randomly oriented tubular filaments undergo a conformational change to more regularly arranged thin filaments, thus causing development of birefringence.

The stalk of the heterotrich ciliate, Stentor coeruleus, shows the ability to undergo rapid contractions with an extreme degree of shortening. Speculations have been made suggesting either that the contractile system is similar to the two-filament system of metazoan muscles or that the contraction is based on a different mechanism. Light and electron microscopic observations have demonstrated the existence of two fibre systems parallel to the longitudinal axis of Stentor, and these two systems have been related to the length changes of the animal. Although the km-fibres were formerly considered the contractile element [16], present evidence favours the view that the myonemes are responsible for the contraction [l, 121. Electron microscopic investigations of the myonemes show a filamentous system different from the well-known actomyosin system. Additional information concerning the orga9-741812

nization of the contractile system can be obtained from observations in polarized Ii&t which can reveal regularly organized stnuctures with the advantage that such observations can be performed on living animals. The present work deals with an analysis of the patterns of birefringence exhibited by Stentor coerulezu under changes in length: and a comparison of these with t structure of the contractile system. MATERIAL

AN

ETHODS

Stentor coeruleus was cultured in flat plastic boxes containing sterilized pond water. Colpidium colpoda was addend as a food supply. The culture was maintained by transferring subcultures into fresh media-m every 4 weeks. Birefringence in Stentor was studied with a Zeiss RP 48 polarizing microscope equipped with a 61/-I 5W illuminator, and photographs were taken on llford HP4 film. Exposure times varied, depending upon the magnification, and generally exceeded 20 sec. When shorter exposure times were required, the polarizing equipment was transferred to a Zeiss photomicro-

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scope II using an HBO 200 W/4 UV lamp as illuminator. To ensure maximum light all filters were omitted during the exposures, thereby reducing the exposure times to less than 1 sec. Polarizing microscope observations were performed on living animals in their culture medium. However, isolated extended stalks could be prepared from EGTA-relaxed animals [6] by cutting away the body part with a needle and transferring the stalk to a microscope slide for direct observations in the polarizing microscope. Extended animals were prepared for electron microscopy according to B. Huang’s method [6] for fixing animals in the extended state using a relaxing medium containing EGTA. Fixation and embedding were performed as described below. Animals were also glycerol extracted in the contracted state and incubated with heavy meromvosin (HMM). Glvcerination of Stators and incubation wi&HMhi were performed according to the procedure of Ishikawa et al. [9]. As a control, animals were incubated in the same salt solution, omitting HMM. Heavy meromyosin was prepared from rabbit skeletal muscle myosin according to the method of Szent-Gyiirgyi [15]. The reactivity of HMM was checked on rabbit skeletal muscle actin [ll] by demonstrating arrowhead formation, using negative staining with uranyl acetate according to Huxley [8]. Following incubation with or without HMM, the Stentors were fixed for 1 h at 4°C with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer pH 7.3. The fixed Stentors were washed twice with the-cacodylate buffer and postfixed for 30 min in 1% 0~0, in 0.067 M Verona1 acetate buffer pH 7.3 at 4°C. After briefly rinsing in buffer the cells were block-stained for 1 h with i % uranyl acetate in water, washed and dehydrated in a graded series of ethanol solutions and subsequently passed through propylene oxide before embedding in Epon. Thin sections were cut on a LKB ultratome (LKB Instruments, Stockholm, Sweden) and stained with uranyl acetate and lead citrate. The sections were observed in a Philips EM 300 electron microscope.

RESULTS The living, extended Stentor coevuleusshowed a prominent birefringence in the stalk and the ciliated membranellar band. Fig. 1 shows 4 extended animals in different orientations photographed with polars crossed. In fig. 1 the polars were oriented so that their axes were parallel to the edges of the figure. It is

seen that the stalk appears most brilliant in the 45” position with respect to the axes of the polars, indicating maximum birefringence. In positions parallel to the axes birefringence in the stalks were extinguished, while the membranellar band remained birefringent. The anterior body segment appeared dark in all positions. Fig. 2 shows a series of photographs taken at intervals during the elongation period. In this figure and all the following figures showing polarization the length axis of the animals were oriented at about the 45” position. Due to the slightly sweeping movements of the animal it was necessary immediately before each exposure to rotate the microscope stage slightly in order to reorientate the length axis of the animal in the 45” position. Shortly after contraction the animal showed almost no birefringence. Approx. 15 set later the animal started to stretch and both the aboral end and the membranellar band acquired some birefringence. As the elongation proceeded the intensity and extent of the birefringence increased considerably and 3 min after beginning full extension and maximum birefringence was achieved. Part of an isolated stalk from an animal in relaxation medium can be seen at higher magnification in fig. 3. The birefringence is found in parallel, close-lying stripes running in the longitudinal direction. With long exposure times, birefringent stripes could also be found in unextended animals as illustrated in fig. 4. In the upper part of the figure the birefringence patterns consist of smooth stripes, while at the lower part, close to the holdfast, the patterns resemble broken lines. Fig. 5 shows the same part of the animal as in fig. 4, but now photo-

Fig. 1. Four extended Stentor coeruleus in different orientations photographed in a polarizing microscope with polars crossed. x 50. Fig. 2. (u-e). A series of photographs taken at intervals during the elongation period, illustrating the development of birefringence in Stentor c. x40. Exptl

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graphed in non-polarized light. The myonemes near the holdfast show the lateral convolutions described by Bannister & Tatchell [l]. Comparison of figs 4 and 5 demonstrates that the myonemes in the convoluted form correspond to the broken birefringent lines while the straight part of the myonemes corresponds to the unbroken birefringent part of the stripes. When complete contraction was induced by electrical stimulation 1131 it was observed that the myonemes straightened and the birefringent lines hereafter appeared unbroken over their entire length. It is of interest to note that significantly longer exposure times were required in order to demonstrate birefringence of the myonemes in the unextended animal. It is thus apparent that the intensity of birefringence of the myonemes in the extended stalk was appreciably greater than myoneme birefringence in the unextended animal. A cross-section of part of a stalk from an extended Stentor is shown in fig. 6. The two fiber systems, the myonemes and the kmfibres, are situated beneath the pellicle. The km-fibres consist of stacks of microtubules and the inset shows that the myonemes consist mainly of thin filaments (3-4 nm) and some 10 nm tubular-like filaments. In fig. 7 the fibre systems are seen longitudinally cut. Fig. 8 shows a myoneme from a contracted,

glycerinated, and HMM incubated Stentor. Due to glycerination the fiber appears rather loose. Many 10 nm tubular-like filaments are seen. No arrowhead formation could be demonstrated. Isolated stalks and segments of stalks in relaxation medium appeared birefringent in polarized light. On addition of Ca2+ they contracted and the birefringence disappeared. This is illustrated in figs 9 and 10 showing an isolated stalk before and after addition of Ca2+, and photographed in non-polarized as well as polarized light. DISCUSSION In 1875 Engelmann [3] reported that the contractile protozoan Stentor coeruleus showed a pronounced birefringence in the extended state and that this was lost upon contraction. Until now, however, the birefringence has not been unambiguously related to the discrete structures in the animal. In the present study it can be seen that the birefringence occurs in stripes and that maximum brilliance is found along the edge of the stalk. Since a number of fibres would appear stacked at the edge giving rise to an increased intensity, it is reasonable to relate the birefringence to one or both of the two fibre systems running longitudinally beneath the pellicle (fig. 6).

Fig. 3. Part of an isolated stalk from Ste~tov c. showing parallel birefringent stripes running in the longitudinal direction. x 515. Figs 4, 5. Aboral end of an unextended Stentov c. photographed in polarized light [4] and in ordinary light IS]. At the lower end, close to the holdfast, the myonemes appear dashed in polarized light, corresponding to the convoluted course seen in ordinary light. x 515. Fig. 6. A cross-section of an extended stalk showing the two subpellicular fibre systems, the km-fibres and the myonemes x 36 000. The inset shows part of a myoneme at higher magnification. The arrow indicates a 10 nm tubular-like filament in cross section. x 160 000. Fig. 7. A longitudinal section of an extended stalk. A km-fibre is seen in the upper part. The myoneme appears in the lower part of the figure. x 93 000. Fig. 8. Part of a myoneme from a contracted, glycerinated and HMM incubated Stentor. Arrows indicate 10 nm tubular-like filaments. x 93 000. Fig. 9. An isolated stalk from Stentor c. in EGTA-medium photographed in (a) ordinary and (b) polarized light. x 140.

E9g. IO. Same stalk as shown in fig. 9 after addition of excess Ca ‘+. Photographed zed light. x 140. Exptl

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134 Kuistensen, Engdahl Nielsen and Rostgaard One of the fibre systems consists of the kmfibres. Earlier observations related this system to the birefringence [4]. These observations were supported by Randall & Jackson [14] although they stated that the km-fibres were displaced from the position in which the birefringence was found. The km-fibres consist of a series of parallel stacks of microtubules. The number of stacks in each km-fibre cross section is small in extended animals, while up to 66 have been found in contracted specimens [l]. This indicates that the stacks are of a fixed length and slide past each other as the body changes its length. If the birefringence were caused by the stacks of microtubules it should be expected that a higher intensity would be seen in contracted animals as the highest number of stacks is found in this state. In fact, the birefringence is maximal in extended animals with only a few stacks of microtubules in each cross section thus indicating that the microtubules do not contribute much to the observed birefringence. Attention is therefore drawn to the other fibre system, the M-bands or myonemes, to which the contraction is attributed. The myonemes are found in the aboral part of the animal, being thicker near the holdfast and tapering towards the adoral end [l]. It is important to note that the birefringence is restricted to the part of the animal where the myonemes are found. Immediately after contraction the myonemes are straight but soon after they exhibit lateral convolutions [I]. As the body begins to extend the myonemes gradually restraighten, while the strong birefringence reappears. In contracted animals weak birefringence was present in stripes which appeared straight immediately after contraction, but soon after became dashed in the posterior end following the convolutions of the myonemes. The fact that the birefringence in the extended animal is found in the same part of the animal as the Exptl

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myonemes, and that it follows changes in the shape of the myonemes in the unextended animal, strongly indicates that it is the myonemes which possess the birefringent properties. Electron microscopic observations of the myonemes have shown two types of microfilaments in both the extended and the contracted state. Huang & Pitelka [7] found that the myonemes in fully extended animals consisted mainly of small bundles of 4-5 nm filaments longitudinally oriented. In the contracted state the majority of filaments were tubular in cross section with an outside diameter of lo-12 nm. These tubular filaments were more randomly oriented and densely packed. Our results are in general agreement with those of Huang & Pitelka although we found the thin filaments to be about 3 nm in diameter. Bannister & Tatchell [2] also found two types of filaments, measuring 3-5 and 8 nm in diameter. In contrast to our results, however, theirs showed the two filament types to be present in approximately the same ratio in both contracted and uncontracted myonemes. The microfilaments of contracted myonemes were closely aggregated, whereas those of uncontracted myonemes were more widely spaced. This discrepancy may, however, be related to the different methods used to relax the animals. In Huang’s study as well as ours the relaxation was accomplished by the Ca2+ complexing agent EGTA, which efficiently causes full extension in Stentov. Birefringence can be related to an alignment of linear elements. Loss of birefringence may result from a disorganization of the regular arrangement. The pronounced birefringence in extended myonemes is most probably due to a highly ordered array of the 3-4 nm microfilaments, and the loss of birefringence on contraction may be correlated with conformational changes of the filament-

ous structures resulting in the more randomly oriented 10 nm tubular-like filaments. A two-filament mechanism is believed to operate in most contractile systems. A prominent feature of this system is that the two types of filament slide past each other without any change in length. The immediate energy source for this type of contraction is adenosinetriphosphate (ATP). In striated muscle and in many other motile cells the two filament types are constituted of actin and myosin, respectively. The presence of actin in a variety of cell types has been demonstrated by its reaction with heavy meromyosin M) (for review see Komnick et al. [lo]) until recently it was believed that the yosin system was ubiquitous. th the fmding that the myonemes in Stentor did not combine with HMM as well as the differences in appearance and dimensions of the microfilaments suggest a contractile system which is different from the actomyosin system. Such a system has been described in some other peritrich ciliates by HoffmannBerhng [.5] and Weis-Fogh & Amos [17]. The spasmoneme in these peritrich ciliates, Iike the contractile myonemes in Stentor, showed positive birefringence in the extended state, and contraction could be mediated by Ca2+ with no requirement for ATP. The shortening in Stentov involves a length reduction of approx. 70% in less than 10 msec. This degree of extremely rapid shortening is special for this type of ciliate and can possibly be described in terms of Ca2+-initiated confosmationai changes of the myoneme microfilaments into the more randomly oriented tubular filaments.

From the present study it appears evident that the contractile system of Sr’entor is located in the myonemes and that the contractile process is God-muscular in character. Further studies an-eplanned with the purpose of investigating the contractile system on the molecular level. The authors wish to thank Gurli Bengtson and K&ten Sjeberg for excellent technicai assist&e. This work was supported by grants from the Danish State Research Foundation and the Tuborg Foundation to J. Rostgaard.

1. Bannister, t H & Tatchel?, E C, J cell sci 3 (1968) 295. 2. - Exptl ceil res 73 (1972) 221. 3. Engelmann, T W, Pfliiger’s arch physiol 1 ! (1875; 432. 4. Faur&Fremiet, E, Rouiiler, C & Gauchery, M, Arch anat microsc morph01 exptl 4.5 (1956) 139. 5. Hoffmann-Berling, H, Biochim biophys acta 27 (1958) 241. 6. Huang, B, J cell biol 47 (1970) 92~ 7. Huang, 3 & Pitelka, D R, J cell biol57 (1973) 704. 8. Huxley, H E, Jmol biol7 (1963) 281. 9. Ishikawa, H, Bischoff, R & Holtzer, 43 (1969) 312. 10. Komnick, H, Stockem, W & Wohl mann, K E, Fortschr zoo1 21 (1972) 1. 31. Mommaerts, W F H M, Methods in mei”icai research (ed J V Warren) vol. 7, p. 1. The Yearbook Publishers, Chicago (1958). 12. Newman, E, Science 177 (1972) 447. 13. Nielsen, L E, Comp biochem physiol 4OA (1971) 639. 14. Randall, J T & jackson, S F, J biophys b&hem cytol4 (1958) 807. 1.5. Szent-GyCrgyi, A G, Arch biochem biophys 42 (1953) 305. 16. Villeneuve-Brachon, S, Arch zoo1 exptl gen 82 (1940) 1. 17. Weis-Fogh, T 8i Amos, W B, Nature 236 (1972j 301. Received July 23, 1973 Revised version received November 13, I973