TENTACLE CONTRACTION IN GLYCERINATED DISCOPHRYA COLLINI AND THE LOCALIZATION OF HMM-BINDING FILAMENTS

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1 J.CellSci. 47, 65-75(1981) 65 Printed in Great Britain Company of Biologists Limited 1981 TENTACLE CONTRACTION IN GLYCERINATED DISCOPHRYA COLLINI AND THE LOCALIZATION OF HMM-BINDING FILAMENTS CAROLE M. HACKNEY AND R. D. BUTLER E.M. Unit, Department of Botany and Zoology, The University, Manchester Mi 3 gpl, England SUMMARY The contractile tentacles of the suctorian Discophrya collini contain a central microtubular axoneme as well as filamentous structures in the cortical epiplasm and in a fibrous collar around the axoneme at the tentacle base. The nature and possible roles of these components has been investigated by the use of reactivatable glycerinated cells. In these a mean tentacle contraction to 70 % of the original length could be achieved by a 5-min treatment with a reaction mixture containing ATP, calcium and magnesium ions, the same treatment giving retraction to 30 % in living cells. Both the microtubules of the axoneme and the filaments of the fibrous collar and epiplasm were present in the glycerinated cells, suggesting that these components consist of large water-insoluble molecules. The addition of heavy meromyosin to whole glycerinated cells resulted in the appearance of nm spaced 'tails' orfilamentsattached to the epiplasmic fibres and the aggregation of 3-6-nm filaments and electron-dense material in the region of the fibrous collar. Neither of these 2 features was apparent after treatment with ATP. It is suggested that actin-like filaments are localized in the region of the fibrous collar and in the epiplasm, and that these are involved in tentacle retraction; whilst the microtubules of the axoneme are concerned with feeding, and play only a cytoskeletal role in the contractile mechanism. INTRODUCTION Suctoria are modified ciliates, characterized by their lack of cilia in the adult stage and the possession of tentacles used for feeding. The long capitate tentacles of Discophrya collini undergo periodic unsynchronized retraction and extension movements, and will retract in response to a variety of mechanical, chemical and electrical stimuli (Curry, 1974; Sabri, 1977; Hackney & Butler, 1978; Hackney, 1979). Their ultrastructure follows the general suctorian pattern, containing a central cylinder (axoneme) of 2 concentric rings of microtubules, the inner one being organized into lamellae (Curry & Butler, 1976). The apical knob is covered by plasma membrane only but the tentacle shaft is covered by a cortex with a fibrous epiplasmic layer continuous with that of the cell body. A short distance (3 fim) below the apical knob, the axoneme microtubules of the outer ring are apparently held together by an electron-dense connective sheath, whilst in the region below the insertion of the shaft into the cell body the axoneme is surrounded by a fibrous collar, fibres from which extend upwards into the periaxonemal space. It has been suggested previously that both microtubules and microfilaments are implicated in tentacle movement. Hauser & Van Eys (1976) concluded that an

2 66 C. M. Hackney and R. D. Butler interaction between microtubules and microfilaments is responsible for contraction in the tentacles of the suctorian Heliophrya erhardi. Earlier, Tucker (1974) had suggested that the contraction of tentacles in Tokophrya might involve the epiplasmic layer. In Discophrya, there is no conclusive evidence which indicates the site of the contractile mechanism. Ultrastructural observations indicate that the microtubules of the axoneme do not generate the motive force for tentacle movements either by sliding or depolymerization (Curry & Butler, 1976). The presence of a fibrous collar region and a thick fibrous epiplasm suggests that functional microfilaments, possibly composed of actin, could be present. Forer & Behnke (1972) state that it seems likely that all motile systems contain actin-like and myosin-like proteins and indeed these are found in a wide range of situations and cell types. As the tentacles of suctorian protozoa have a contractile structure containing microtubules and possibly microfilaments, they represent suitable material in which the role of these 2 components in cell movement may be studied. Since glycerination leaves the water-insoluble parts of contractile systems intact (Nagai & Kamiya, 1966) and the reactivation of a glycerinated system indicates the continued presence of the contractile molecules, glycerinated cells have been used here to investigate the possible role of the filaments found in the fibrous collar region and epiplasmic layer. This paper also describes ultrastructural studies of the glycerinated cell. Glycerination has also allowed attempts to identify the nature of the microfilaments by the addition of heavy meromyosin (HMM) which is known to bind F-actin (Huxley, 1963; Ishikawa, Bischoff & Holtzer, 1969; Pollard & Korn, 1971; Behnke, Kristensen & Nielsen, 1971a, b). These results are discussed in relation to possible contractile mechanisms and the wider implications of the presence of actin are considered. MATERIALS AND METHODS Discophrya collini (obtained from the Cambridge Culture Collection) was cultured attached to short lengths of silk thread or coverslip fragments in 5-cm Petri dishes in an inorganic medium (Stewart, 1972), fed with Colpidium every 3 or 4 days and subcultured every 2 weeks. Cultures could be examined in situ using x 10 and x 40 water-immersion lenses. For experimental observations, the small pieces of silk or coverslips with attached specimens were mounted in culture medium or test solution and observed with bright-field, positive or negative phase-contrast or Nomarski interference optics. For transmission electron microscopy (TEM), specimens were fixed in a solution of 12-5 % glutaraldehyde in 1/30 M Sorensen's mixed phosphate buffer (ph 72) for 30 s, followed by the addition of an equal volume of 1 % OsO 4. The material was dehydrated and embedded in Spurr resin, and thin sections cut using glass knives. Sections were stained for 7-10 min in 25% uranyl acetate dissolved in 66% ethanol followed by lead citrate (Reynolds, 1963). Sections were examined on an AEI EM. 802 electron microscope. For scanning electron microscopy (SEM), a method for fixation based on that of Henk& Paulin (1977) was employed. Discophrya attached to coverslip fragments were fixed in a mixture of OsO 4 and HgCl a (Parducz, 1955), dehydrated and subjected to critical-point drying from 100% ethanol using liquid CO a for the final transition at 1200 lb in.~ s (83 x io 3 kn m" 2 ), 32 C. The mounted specimens were coated with gold in an Edwards sputter coater and examined in a Cambridge Si 50 electron microscope operated at 20 kv. Two methods of glycerination were employed. Method A consisted of immersing cells in a solution of 50% v/v glycerol, 10% DMSO in 5 mm Sorensen's phosphate buffer (ph 6-8), 5 m\i MgCl 2 and EGTA for 2 h at 40 C (Hauser & Van Eys, 1976). In method B, cells were

3 Tentacle contraction in Discophrya 67 placed in a ulyccrol standard salt solution (G/SS) for 3 days at room temperature (Forer & Behnke, 1972). The G/SS consisted of a 1:1 mixture of pure glycerol and standard salt solution (SS). The SS contained final concentrations of 50 mm KC1, 5 mm MgCL and 6 mm Sorenscn's phosphate buffer at a final ph of 70. After either extraction procedure cells were stored at 4 C. Cells were placed in drops of a reaction mixture containing 30 mm ATP (disodium salt) in 10 mm imidazolc, 5 mm sodium azide (NaNj), 17-5 mm MgCI 2, 5 mm CaClj and EGTA (ph 70) (Hauser& Van Eys, 1976). Living cells were also treated with the reaction mixture as controls. Heavy meromyosin (HMM) supplied by Dr A. Curry and Dr D. Woolley, Withington Hospital, Manchester, was tested for ATPase activity before use by the measurement of inorganic phosphate released according to the methods of Sumner (1944) and Lowry, Passoneau, Hasselburger & Schultz (1964). It released 9-16 % P, from 1 mm ATP at a concentration 1 mg/ml after 30-min incubation at room temperature (Mann, 1977). For cell treatment, HMM was mixed with SS (1-2 mg HMM/ml) and method B-glycerinatedcellsweretransferred to, and stored in, this solution for 24 hat 4 C.Glycerinated cells were fixed and embedded without HMM as controls. Some of the HMM-treated cells were placed in ATP/SS which contained 80 mm disodium adenosine-s'-triphosphate (Sigma) at a final ph of 68 for 5 min at room temperature before fixing and embedding. RESULTS Glycerination method A caused bending of the tentacles, and the cytoplasm became highly vacuolated in appearance in comparison with that in living cells (Figs, i, 2). In over 50 cells examined, some of the tentacles of each had become retracted and were wrinkled in appearance. Such tentacle distortions made the use of these cells to determine any response to the retraction mixture impracticable. In a similar number of cells, method B, on the other hand, gave cells with mainly straight tentacles (Figs. 3, 4), although it produced a diffuse appearance of the cell cytoplasm, in which the Spheroid nucleus was often more obvious than in intact cells (Figs. 1, 3). With a mean length of 19 /tm (from 25 cells), these tentacles were shorter than those in living cells of the same age (25 /tm) but they were not wrinkled. In a few tentacles expansion of the tip region occurred giving a 'balloon-like' appearance to the knob. The overall preservation of tentacle morphology is confirmed by SEM; comparison with unglycerinated tentacles (Fig. 5) showed remarkably little damage to the cell surface (Fig. 6B), apart from the tentacle tip which showed some slight swelling and disruption (Fig. 6A). It is evident that the tentacles from method B are considerably more normal in appearance than those produced by method A. Tentacle retraction could be stimulated in the cells produced by method B by the addition of the reaction mixture which contained ATP, Ca ions and Mg ions at those concentrations found to be effective in Heliophrya erhardi (Hauser & Van Eys, 1976) (Figs. 7, 8). After 5-min treatment, the tentacles showed a mean contraction to 70% of the original length (18-89/4111 to 13-5/4111 in 25 cells), some of the tentacles also displaying a bending reaction. In living cells, the same treatment produced a mean retraction to 30% of the original length (27-52/(111 to 8-56/tm in 25 cells). A TEM study of the method B-glycerinated cells showed the cytoplasm to be very disrupted and diffuse in comparison to that of untreated cells (Figs. 9, 10). The mitochondria, rough endoplasmic reticulum and cytoplasmic vesicles were less numerous, those remaining being swollen, with ill-defined limiting membranes. The cortex appeared less affected, becoming more fibrous in appearance with 3-6-nm

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5 Tentacle contraction in Discophrya 69 diameter filaments. The microtubules of the tentacle axoneme were still present but lacking in definition compared with those seen in untreated cells (Figs. 11, 12). In the region of the fibrous collar 3-6-nm filaments could also be seen and were associated with more electron-dense material (Fig. 12). Incubation of whole glycerinated cells with HMM resulted in 2 main ultrastructural changes. Some of the epiplasmic filaments had thin fibres or 'tails' attached to them nm apart (Fig. 13). Also the fibrous collar region appeared to be condensed but still contained filamentous material (Fig. 15). The microtubules of the tentacles were not apparently labelled or disrupted in any way by the HMM treatment, appearing similar to the microtubules seen in the glycerinated cells. If HMM-incubated cells were treated with ATP/SS before fixation and embedding, their ultrastructure appeared similar to that of the glycerinated controls (Fig. 14), neither the HMMlabelling of the cortex nor the condensation of the fibrous collar material, being apparent in any section examined. DISCUSSION In attempting to develop a glycerination technique for the tentacles of Discophrya collini, the method developed by Hauser & Van Eys (1976) for another suctorian protozoan Heliophrya seemed most likely to be suitable. In Heliophrya this technique appears to have caused some bending or retraction of the tentacles. In Discophrya, the degree of tentacle distortion seemed more pronounced and was such that any retraction caused by the reaction mixture would not be distinguishable from that caused by this method of glycerination. In addition, of course, this distortion could well influence the relative positions of the contractile system components to be examined by TEM. The method of glycerination devised by Forer & Behnke (1972) for cranefly spermatids was more successful in producing straight unwrinkled tentacles. The damage to the tentacle tip may well have been due to much greater penetration of this area which is covered by a plasma membrane only. The post-glycerination disruption of the cytoplasm which was observed has been found also in other systems such as Chaos (Comly, 1973) and in glycerinated spermatids (Forer & Behnke, 1972). This latter system reacted dramatically to the reaction mixture containing ATP. Retraction and wrinkling of the tentacles occurred, and though these changes were Fig. 1. Untreated living cell showing long straight capitate tentacle (t), contractile vacuole (cv) and macronucleus (mn). Nomarski interference, x 620. Fig. 2. Cell glycerinated by method A (see text) showing short distorted tentacles and apparently vacuolated cytoplasm. Nomarski interference, x 500. Figs. 3, 4. Cells glycerinated by method B (see text). The tentacles remain straight and relatively extended but in some instances show expanded tips (arrowed). Fig. 3, x 630; Fig. 4, x 460. Fig. 5. Tentacle of untreated cell. The protuberances on the apical knob mark the position of the underlying haptocysts (ha). SEM. x Fig. 6. Tentacle of cell glycerinated by method B. A. Detail of tentacle tip showing some disruption of apical knob. SEM, x B. Note tentacle shaft remains smooth, comparable to that in the untreated control. SEM, x

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7 Tentacle contraction in Discophrya 71 not as marked as in living cells, they nevertheless indicated the continued presence of a reactivatable contractile system in the tentacle, suggesting the involvement of large water-insoluble molecules. It is relevant that both the microtubules and the fibres of the cortex and collar region were still apparent after glycerination. The reaction of HMM in these regions suggests the possible presence of actin-like protein, especially since this interaction is no longer apparent after treatment with ATP, as HMM is known to bind F-actin (Huxley, 1963) and because ATP is known to prevent this binding (Huxley, 1963; Ishikawa et al. 1969; Behnke et al ; Forer & Behnke, 1972). Although the characteristic arrowheads with regular 36-nm spacing on actin fibrils seen in other glycerinated systems (Ishikawa et al. 1969; Pollard & Korn, 1971) were not observed, 'tails' nm apart were seen in the cortex. This may, of course, indicate the absence of actin or alternatively be due to the fact that bundles of filaments in varying orientations occur, or are arranged in a complicated 3-dimensional network thus reducing the possibility of clearly demonstrated 'arrowheads' in thin sections of whole cells (Forer & Behnke, 1972; Buckley & Raju, 1976). It may be that cortical actin-like filaments are present in other suctorians. Similar HMM-decorated filaments have been seen in the tentacle cortex of Dendrocometes paradoxus, but could not be demonstrated in Tokophrya infusionum (Curry & Woolley, 1975). If actin-like fibres are localized in the fibrous cortex region, it would seem likely that this region is indeed involved in tentacle contraction in suctoria (Tucker, 1974; Spoon, Chapman, Cheng & Zane, 1976). Although the major function of the microtubule system of the axoneme may be in feeding (Curry & Butler,' 1976) it may provide also a rigid support against which the cortex might act. The axoneme does not appear to be attached to any structure other than the cortex and then only at the tentacle tip, so it is difficult to see how it alone could produce the motive force necessary for contraction, especially since little evidence has been found for microtubule rearrangement (Curry & Butler, 1976). In the suctorian Dendrocometes paradoxus, a slight decrease in helicity occurs in the microtubules of the axoneme during tentacle retraction, suggesting that the axoneme is pulled down into the cytoplasm (Geldard, 1977). In Discophrya it seems more likely that contractile filaments in the epiplasm produce the motive force necessary for contraction, the attachment of the axoneme to the cortex at the tentacle tip (Curry & Butler, 1976) providing a strengthened region tending to force the axoneme down into the cell body. The fibrous collar region might then act as an anchor for the axoneme, and also provide antagonistic forces to the filaments of the epiplasmic layer. It could possibly force the axoneme upwards again Figs. 7, 8. Cells glycerinated by method B followed by treatment with reaction mixture (see text). Addition of reaction mixture has resulted in tentacle retraction and some distortion (cf. Figs. 3, 4). Normarski interference. Fig. 7, X630; Fig. 8, X57. Fig. 9. TEM section through cortical region in body of untreated cell, showing layered epiplasm (epi) and dense cytoplasm, x Fig. 10. Section through cortical region in body of cell glycerinated by method B. Note fibrous appearance of epiplasm, the diffuse appearance of the mitochondria (m) and relative paucity of cytoplasmic components, x

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9 Tentacle contraction in Discophrya 73 4 Fig. 15. Section through fibrous collar region of cell glycerinated by method B and treated with HMM. The region of the fibrous collar contains electron-dense material with 3-6-nm fibres (arrowed). The axoneme microtubules are still evident though less distinct than in unglycerinated controls, x Fig. 11. Vertical section through point of insertion of tentacle into body of untreated cell. The microtubules (mt) of the tentacle axoneme (ax) pass into the region of the fibrous collar (Jc). x Fig. 12. Section through tentacle insertion region in cell glycerinated by method B. Note the continued presence of axoneme microtubules (mt) and components of the fibrous collar (Jc). x Fig. 13. Cell glycerinated by method B and treated with HMM. A. The epiplasm contains fibrous components (arrowed), x B. Detail from afibrouscomponent in A with 'tails' (arrowed) at nmspacing, x Fig. 14. Cortex of cell glycerinated by method B treated with HMM followed by ATP. The epiplasm shows no fibrous components, x

10 74 C. M. Hackney and R. D. Butler by the activity of its filaments pushing cytoplasm and microtubules up the tentacle shaft. In spite of a thorough search no evidence has been found for bundles or networks of microfilaments higher up the tentacles in the periaxonemal space, as have been reported in Heliophrya by Spoon et al. (1976) and by Hauser & Van Eys (1976). This may be due to a difference in fixation procedure but it seems more likely, in view of its poor response to the Heliophrya glycerination method, that the system in Discophrya is quite different. REFERENCES BEHNKE, O., KRISTENSEN, B. I. & NIELSEN, L. E. (1971 a). Electron microscopical identification of platelet contractile proteins: In Platelet Aggregation (ed. J. Caen), pp Paris: Masson & Cie. BEHNKE, O., KRISTENSEN, B. I. & NIELSEN, L. E. (19716). Electron microscopical observations on actinoid and myosinoid filaments in blood platelets. J. Ultrastruct. Res. 37, BUCKLEY, I. K. & RAJU, T. R. (1976). Form and distribution of actin and myosin in non-muscle cells- a study using cultured chick embryo fibrolasts.j. Microscopy 107, COMLY, L. T. (1973). Microfilaments in Chaos carolinensis. Membrane association, distribution and heavy meromyosin binding in the glycerinated cell. J. Cell Biol. 58, CURRY, A. (1974). A Cytological Study of Some Suctorian Protozoa. Ph.D. Thesis, University of Manchester. CURRY, A. & BUTLER, R. D. (1976). The ultrastructure, function and morphogenesis of the tentacles in Discophrya sp. (Suctorida) Cileatea. J. Ultrastruct. Res. 56, CuRRY, A. & WOOLLEY, D. E. (197s). The detection of actin-like proteins in some protozoa using heavy meromyosin. J. Protozool. X2, 50A (Abstr.). FORER, A. & BEHNKE, O. (1972). An actin-like component in sperm tails of a crane-fly (Nephrotoma stituralis Loew).J. CellSci. 11, GELDARD, B. (1977). Cytological and Physiological Studies of Dendrocometes paradoxus (Stein). Ph.D. Thesis, University of Manchester. HACKNEY, C. M. (1979). An Ultrastructural and Experimental Study of the Contractile System in the Tentacles of Discophrya collini (Root). Ph.D. Thesis, University of Manchester. HACKNEY, C. M. & BUTLER, R. D. (1978). The tentacles of Discophrya sp. used as an experimental contractile system. J. Protozool. 25, 6B (Abstr.). HAUSER, M. & VAN EYS, H. (1976). Microtubules and associated microfilaments in the tentacles of the suctorian Heliophrya erliardi Matthes. J. Cell Sci. 20, HENK, W. G. & PAULIN, J. J. (1977). Scanning electron microscopy of budding and metamorphosis in Discophrya collini. J. Protozool. 24, HUXLEY, H. E. (1963). Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. molec. Biol. 7, ISHIKAWA, H., BISCHOFF, R. & HOLTZER, H. (1969). Formation of arrowhead complexes with HMMin a variety of cell types. J. Cell Biol. 43, LOWRY, O. H., PASSONEAU, J. V., HASSELBURGER, F. X. & SCHULTZ, D. W. (1964). Effect of ischemia on known substrates and cofactors of the glycolytic pathways in brain. J. biol. Chem MANN, A. F. (1977). Some Regulatory Aspects of Nitrate Assimilation in Spinach Leaves. Ph.D. Thesis, University of Bristol. NAGAI, R. & KAMIYA, N. (1966). Movement of the myxocete Plasmodium. II. Electron microscope studies of fibrillar structures in the plasmodium. Proc.Jap. Acad. 42, PARDUCZ, B. (1955). Ciliary movement and co-ordination in ciliates. Int. Rev. Cytol. 21, POLLARD, T. D. & KORN, E. D. (1971). Filaments of Amoeba proteus. II. Binding of heavy meromyosin by thin filaments in motile cytoplasmic extracts. J. Cell Bid. 48, REYNOLDS, S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 39, SABRI, A. W. (1977). Cytological Studies of Suctorian Protozoans. Ph.D. Thesis, University of Manchester.

11 Tentacle contraction in Discophrya 75 SPOON, D. M., CHAPMAN, G. B., CHENG, R. A. & ZANE, S. F. (1976). Observations on the behaviour and feeding mechanisms of the suctorian Heliophrya erhardi (Rieder) Matthes preying on Paramecium. Trans. Am. microsc. Soc. 95, STEWART, G. R. (1972). The regulation of nitrate reductase level in Lemna minor Y,.J. exp. Bot. 23, SUMNER, J. B. (1944). A method for the colorimetric determination of phosphorous. Science, N. Y. 100, TUCKER, J. B. (r974). Microtubule arms and cytoplasmic streaming and microtubule bending and stretching of intertubulc links in the feeding tentacle of the suctorian ciliate Tokophrya. J. Cell Bio!. 62, (Received 25 February 1980)

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