Cytoskeletal proteins of the cell surface in Tetrahymena

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Experimental Cell Research 123 (1979) 31 l-320

CYTOSKELETAL

PROTEINS

SURFACE I. Identification NORMAN E. WILLIAMS, Department

of Zoology,

OF THE CELL

IN TETRAHYMENA

and Localization

of Major Proteins

PIERRE E. VAUDAUX’ University

and LARS SKRIVER’

of Iowa, Iowa City, IO 52242, USA

SUMMARY Isolated pellicles (cell ‘ghosts’) have been prepared from Tetrahymena thermophila strain B by two different methods. Using differential solubilization in combination with polyacrylamide gel electrophoresis and electron microscopy, we have tentatively identified the major proteins found in the surface-associated cytoskeleton. The ‘epiplasm’, a continuous layer of fibrous material found just beneath the surface membranes, appears to contain two major proteins. The smaller of the two (mol. wt 122000 D) is believed to be present throughout the layer, whereas the larger protein (mol. wt 145000 D) appears to be localized in the regions where ciliary basal bodies connect to the epiplasmic layer and to surface membranes. Evidence is presented which suggests that actin may also be present in this structure. Tubulin has been isolated from the cytosol of Tetruhymena and compared with cytoskeletal tubulin and porcine brain tubulin. A major protein of mol. wt 250000 D which is found in Tetrahymena pellicles appears to be the major component of kinetodesmal fibers (striated elements which attach to the ciliary basal bodies).

It has long been appreciated that the cell surface in ciliated protozoa represents an unusually high level of structural differentiation. The complex surface structure has been of continuing interest for many years, and modern studies of cortical morphogenesis and cortical inheritance have been especially rewarding (e.g. [l-3]). The molecular bases of the important biological processes manifest in the cell surface of ciliates, however, remain largely unknown. One approach to this problem is to isolate and characterize the molecular components of ciliate surface membranes and related structures. Although a substantial amount of information is available on the lipid composition of surface membrane in ciliates [4], our understanding of surface-associated proteins is more rudimentary.

In the present study we have isolated pellicles (cell ‘ghosts’) from Tetruhymena thermophila and studied the major proteins of the cytoskeletal elements associated with the inner side of the surface membrane system. The main result is the isolation and characterization of the major molecular components of the ‘epiplasm’ [5], which is probably the ciliate-equivalent of the spectrin meshwork found on the inner surfaces of erythrocyte membranes. Our findings suggest that the epiplasm consists of two relatively high molecular weight proteins, probably in association with actin. The two ’ Present address: Division des Maladies Infectieuses, Deoartment de Medecine. Honital Cantonal, Gentve, . Switzerland. 2 Present address: Zoophysiological Laboratory C, Atigtist Krogh Institute, Copenhagen, Denmark. Exp Cell Res 123 (1979)

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high molecular weight proteins are present in nearly equal amounts, but have different spatial distributions within the cytoskeleton. It is suggested that the epiplasm may be responsible for the controlled migration and positioning of surface-related organelles in ciliated protozoa. MATERIALS

AND METHODS

Tetruhymena thermophila strain B was grown at 25°C in 1% nroteose-eentone suonlemented with 0.5% yeast exiract. Fo; pellicle isbiations, the cells were grown in 200 ml cultures to a density of from l-3 x lo5 cells/ml. Between 2 and 4x 10’ cells were used for each isolation. Pellicles without membranes were isolated according to the detergent method of Vaudaux [6]. Pellicles with membranes were isolated using the non-detergent method of Nozawa & Thompson [7, 81. Both types of isolated pellicles were extracted at low ionic strength in 0.1 mM EDTA, 1 mM Tris-HCl, pH 8.3 at 0°C. In each case the pellet from one isolation was suspended in l-5 ml and dialysed overnight against 100 vol of the low ionic streneth buffer. The insoluble residues were then collected Thy centrifuging for 20 min at 25 000 g, and either solubilized for SDS polyacrylamide gel electrophoresis or prepared for electron microscopy. The soluble fractions (25 000 g supematants) were concentrated to 250 ~1 by dialysis against 2 M sucrose, then solubilized by adding an equal volume of SDS solubilizing solution at double strength. Protein in the soluble fraction was polymerized in vitro by the addition of 0.1 M KCl. Actin was prepared from beef heart muscle using the procedure described by Forer [9]. The preparation was tested by HMM decoration of F-actin filaments in vitro and visualization bv electron microscopy. Heart muscle actin was solubihzed and used as a marker in polyacrylamide gel electrophoresis studies of Tetrahymena pellicle proteins. A direct attempt to isolate actin from Tetrahymena pellicles was made by preparing an acetone powder of the pooled pellicles from three isolations, then applying the standard procedure described for the isolation of muscle actin [9]. Tubulin was isolated from the cytosol of Tetrahymena and compared with cytoskeletal tubulin from Tetrahymena and with porcine brain tubulin. Cytosol tubulin was prepared using a modification of the Marantz et al. method [32] developed by Dr Gordon E. Stone, University of Denver (personal communication). Approx. 106 cells were taken up in 5 ml of GTP buffer (0.1 mM GTP, 0.01 M MgC&, 0.01 M KH,PG,, pH 6.5) and homogenized at 0°C for 2 min in a Sorvall Omnimixer at maximum setting. The brei was centrifuged for 30 min in the cold at 25 000 g. The supematant was then diluted to 6 ml with GTP buffer and clarified by centrifugation at 100000 g for 1 h at 0°C. Next 4.5 ml of the high speed supematant was pipetted to a 15 ml centrifuge tube and 0.5 ml of GTP buffer containing 5 mg vinblastine sulfate (VLB) was Exp Cell Res 123(1979)

added. After mixing, the preparation was allowed to stand overnight in the refrigerator. A precipitate ordinarily appears within 1 h and may be centrifuged out at 25000 g the next day. The VLB precipitate was solubilized and subjected to SDS polyacrylamide gel electrophoresis along with cilia, peilicies, and porcine brain tubulin. Brain tubulin purified by phosphocellulose chromatography was kindly provided by Dr Marc Kirschner. Tetrahymena cilia were isolated according to the method of Gibbons [lo]. Samples to be analysed by SDS polyacrylamide gel electrophoresis were treated according to the procedure of Laemmli [ 111. The proteins were separated in 7.5% tube gels either 6.5 or 9.5 cm long, and gels were run at 3 mfiliampsltube. The gels were stained 3 h in 0.25 % Coomassie brilliant blue solution in methanol : water: acetic acid (5 : 5 : I), destained by diffusion in the same solution minus the dye, and stored in 7% acetic acid. Optical density profiles were obtained by scanning at a wavelength of 550 nm in a Beckman spectrophotometer with an accessory gel carriage. Samples were run in urea gels as described earlier

[121.

Pellicle fractions to be analysed by electron microscopy after thin sectioning were fixed in a freshly made mixture of glutaraldehyde and osmium tetroxide as described previously [12]. The samples were then brought to 50 % ethanol, centrifuged to make a pellet, and embedded in 2% agar. Cut agar blocks were subsequently dehydrated and embedded in Epon 812 in the usual manner [ 131.Sections were cut on a Reichert Om U2 ultramicrotome and observed with a Philips EM 300 electron microscope. Extracted pellicles were also analysed by negative staining. A drop of the susnension was added to a coated grid, and most of the huid was withdrawn immediate& with a filter paper. To the remaining film was added a drop of 1% aqueous uranyl acetate, and this was immediately withdrawn similarly. The final film was allowed to air-dry and the grids were examined in the electron microscope. Preparations of band C polymerized in vitro were also examined by electron microscopy using this procedure.

RESULTS Characterization

of isolated pellicles

The major structures present in Tetrahymena pellicles isolated by the detergent method are illustrated in fig. 1. The pellicles are held together in the absence of membranes by the epiplasm, a continuous layer of fibrogranular material found just below the surface membranes in intact cells. Attached to this cytoskeletal framework are several types of microtubular structures. One of these, the ciliary basal body, has an attached rootlet structure called the kineto-

Cytoskeletal proteins in Tetrahymena

313

Fig. 1. Electron micrograph of an individual Terrahymena pellicle prepared by the detergent method of Vaudaux [6]. Surface membranes are removed by this procedure; the resulting cell ‘ghosts’ are essentially the surface membrane-associated cytoskeletal elements present in living cells. The major structural com-

ponents are~the continuous layer of epiplasm (E), attached ciliarv basal bodies (BB), microtubule ribbons (MI?), and the kinetodesmal fibers (KD). Note that one set of microtubule ribbons attaches to the outside of the epiplasm whereas others attach to the inside.

desmal fiber. The remaining microtubules are organized into several sets of ribbons which attach to the epiplasm, some on the inside and others on the outside. The major proteins (bands) found in SDS polyacrylamide gels of isolated Tetrahymena pellicles are seen in fig. 2. There is a great deal of tubulin in the pellicles (fig. 2, band F), as expected from the ultrastruc-

ture. This protein comigrates with tubulin isolated from cilia as shown in fig. 2, and it represents approx. 38 % of the total protein resolved in SDS gels of isolated pellicles. The relatively minor proteins D and E have been identified previously as belonging to the oral structures [6]. This leaves the major proteins A, B, and C to account for the major non-microtubular components of

x36400.

Exp Cell Res 123 ilY79)

314 Williams, Vaudaux and Skriver

Fig. 2. Major proteins of the Z’etrahymena surface

cytoskeleton as resolved by SDS polyacrylamide gel electrophoresis (left gel). The major bands are labeled A-F. The doublet band F is tubulin, and this comigrates with tubulin from isolated cilia (right gel). The major band at the top of the gel on the right is ciliary dynein.

isolated pellicles. Band C represents approx . 12% of the protein seen in these gels; A is near 7 %, and B is approx. 8% of the total. Identification A, B, and C

of the major proteins

Studies involving differential solubilization were carried out in an attempt to determine the relationship between the three unidentitied proteins and pellicular structures. In the present study we extracted the isolated pellicles at low ionic strength using 0.1 mM EDTA in 1 mM Tris-HCl, pH 8.3, at 0°C. This was done primarily because this treatment has been shown to extract a major structural protein, spectrin, from the memExpCrUResI23(1979)

brane-associated cytoskeleton in red blood cells. The results of extracting detergentisolated Tetrahymena pellicles at low ionic strength are illustrated in fig. 3. Densitometric scans of SDS polyacrylamide gels containing whole pellicles, extracted pellicles, and the proteins solubilized by this treatment are presented in fig. 3. It can be seen that bands A and C were solubilized to a large extent, whereas band B remained primarily with the insoluble residue. The residue was then examined microscopically to see which structures persisted in this fraction. The results of both light and electron microscopical examinations of the insoluble residue suggested that the integrity of the pellicles had been lost due to dissolution of the epiplasm. The kinetodesmal fibers were also absent from these preparations. The only pellicular structures which could be found in this fraction were microtubules, and pellicular rings with attached terminal plates. The latter structural complex, shown in fig. 5, represents the attachment sites in the cytoskeleton for the ciliary basal bodies. Band B, the only major protein in this fraction other than tubulin, must therefore represent at least some part of this attachment complex. The results suggest further that the epiplasmic layer may be composed largely of either band A or band C, with the excluded protein probably attributable to the kinetodesmal fiber. Pellicles isolated by the non-detergent method of Nozawa & Thompson [7] were also extracted at low ionic strength, primarily to confirm the solubilization of the epiplasmic layer. Both surface and alveolar membranes are retained in pellicles isolated by this method. It was expected that these pellicles would maintain their integrity in the absence of epiplasm, thus permitting confirmation of the loss of epiplasm by elec-

Cytoskeletal

tron microscopic observation of thin sections. The epiplasm is seen just below the inner alveolar membrane in thin sections of these pellicles (fig. 7). Sections through pellicles of this type after extracting at low ionic strength clearly show that the epiplasm is removed by this procedure (fig. 8). Kinetodesmal fibers were found in pellicles isolated by the non-detergent method after they had been extracted with TrisEDTA (fig. 6). This was somewhat unexpected, because extracted pellicles prepared using the detergent method showed no kinetodesmal fibers. The effect of low salt on the major proteins of ‘non-detergent’ pellicles was then analysed by polyacrylamide gel electrophoresis. The results, presented in fig. 4, show that only band C was extracted from these pellicles, whereas both bands A and B remained primarily in the insoluble residue. The retention of band A in the insoluble fraction thus corresponds with the presence of kinetodesmal fibers in this preparation. The concomitant loss of epiplasm and band C from these pellicles also suggests that band C is the major molecular component of this structure. The extraction of a relatively high molecular weight protein from the librogranular cytoskeleton beneath the surface membrane of Tetrahymena suggests some similarity to the extraction of spectrin from red blood cell ghosts. Too see if further evidence for this similarity could be obtained, we added KC1 to solubilized protein C from Tetrahymena and looked for its assembly into fibers. Potassium chloride was added to a final concentration of 0.1 M, samples were spotted on coated grids, then stained with 1% uranyl acetate. Examination of these preparations in the electron microscope showed the appearance of fibrous networks not seen in control preparations without added KCl. This fibrous network,

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proteins in Tetrahymena 4

c A

LIL i

Fig. 3. Extraction of Tetrahymena pellicles prepared with the detergent method using low salt (Tris-EDTA). Densitometric scans of the region of SDS polyacrylamide gels containing the cytoskeletal proteins, A, B, and C are presented for intact pellicles (top), extracted pellicles (center), and the proteins extracted by this procedure (bortom). Although proteins A and C are largely solubilized, protein B remains primarily with the insoluble residue. Fig. 4. Extraction of Tefrahymena pellicles prepared by the non-detergent method of Nozawa & Thompson [7] using low salt (Tris-EDTA). Densitometric scans of the region of SDS polyacrylamide gels containing the cytoskeletal proteins A, B, and C are presented for intact pellicles (top), extracted pellicles (center), and the proteins extracted from these pellicles (borrom). The soluble fraction primarily contains the single protein C.

shown in fig. 9, is morphologically similar to the spectrin network polymerized in vitro by Tilney & Detmers [ 141. Evidence for actin in Tetrahymena pellicles

Actin has been found associated with the cell surface in a number of cell types, and it is present together with spectrin in red blood cell ghosts [14]. In the present study we prepared actin from vertebrate muscle and added it to solubilized pellicles isolated by both detergent and non-detergent methods. Samples of pellicles with and without added actin were then compared using both urea and SDS polyacrylamide gels. It was found that pellicles contain a protein band which comigrates with muscle Exp Cell Res 123 (1979)

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actin in these two electrophoretic systems. Fig. 10 shows the results obtained using SDS gels. We then attempted to isolate actin from pellicles and polyermize it in vitro with the standard methods used for vertebrate muscle. This attempt was unsuccessful. It is possible that the isolation of Tetrahymena actin may present special problems which will require the modification of standard procedures. Until this is accomplished, the co-migration of a pellicular protein with muscle actin during electrophoresis can only be regarded as suggestive evidence for the presence of actin in Tetrahymena pellicles. Tetrahymena

tubulin

The metabolic studies described in the companion paper [33] make use of Tetrahymena tubulin isolated from the cytosol by treating the 100000 g supernatant with vinblastine sulfate and separating the precipitated proteins by SDS polyacrylamide gel electrophoresis. In the present study, the VLB precipitate was compared with cilia in SDS gels to show that the major protein recovered from the cytosol using this procedure co-migrates with ciliary tubulin (fig. 11). Densitometric scans suggest that about 60% of the total protein in the VLB precipitate is tubulin. Tetrahymena tubulin was further compared with porcine brain tubulin in this experiment. As seen in fig. 11, Tetrahymena tubulin did not comigrate precisely with porcine brain tubulin in SDS polyacrylamide gels. We found that brain tubulin ran as a single band when the running buffer was pH 8.6, and that this band comigrated with the upper band of the two seen in Tetrahymena tubulin under these conditions. When the running buffer was pH 8.3, brain tubulin ran as a pair of bands whereas Tetrahymena tubulin ran as a single band which comigrated with the Exp Cell Res 123 (1979)

smaller of the two brain tubulin polypeptides. The molecular basis for the different behaviors of Tetrahymena and brain tubulin in SDS polyacrylamide gel electrophoresis remains to be discovered. This peculiarity of Tetrahymena tubulin has also been noted by Maekawa & Sakai [ 151. DISCUSSION Extraction of cell ghosts at low ionic strength together with SDS polyacrylamide gel electrophoresis and electron microscopy has led to a tentative identification of the major molecular components of the surfaceassociated cytoskeleton in Tetrahymena. Band C, the second-most predominant band in SDS gels of isolated pellicles, is believed to be the major component of the continuous layer of epiplasm found beneath the surface membranes in this cell. Band C was removed from both types of pellicle preparations with a corresponding loss of this layer as determined by electron micro-

Fig. 5. Negatively-stained

preparation of the major structural element (in addition to microtubules) found in the residue of detergent-prepared pellicles extracted with Tris-EDTA. The pellicles appear to dissolve, leaving free circumciliary rings (arrow) containing basal body terminal plates, the-latter sometimes with attached basal body microtubules. The terminal plates often dislodge during preparation for electron microscopy as seen here. The small circular ring to the right in the photograph is the opening of a surface invagination in ciliates called the ‘parasomal sac’. ~95 400. Fig. 6. Negatively stained preparation of microtubules (M) and kinetodesmal fibers (KD) remaining after pellicles isolated by the non-detergent method were extracted bv low salt (Tris-EDTA). x98 400. Fig. 7. Thin section through a.pellicle isolated using the non-detergent method of Nozawa & Thompson [7]. Beneath the outer membrane (rap) lies a system of flattened alveolar membranes. The epiplasmic layer of the cytoskeieton lies just beneath the inner membrane of the alveoli (arrows). x%000. Fig. 8. Thin section through a pellicle isolated with the non-detergent method after being extracted with TrisEDTA. Although the outer and alveolar membranes persist, the epiplasmic layer is removed by this treatment (arrows; compare with fig. 7). x 150000.

Cytoskeletal

21-791813

proteins in Tetrahymena

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Fig. 9. Fibers polymerized in vitro by making a solution of the major epiplasmic protein 0.1 M in KCI. x 145000.

scopy. Further, band C was the only protein solubilized to a significant degree when pellicles isolated by the non-detergent method were extracted with low salt. Addition of 0.1 M KC1 to this solution resulted in the precipitation of a fibrous network which is morphologically very similar to polymerized spectrin from red blood cells. The molecular weight of band C has been determined previously and found to be about 122000 D [16]. This is much lower than the molecular weight of red blood cell spectrin (>200000 D). Nevertheless, the Tetrahymena protein is apparently the major element in a structure comparable to the one which embodies spectrin in erythrocytes; both proteins are soluble at low ionic strength, and both polymerize into a tibrous network of specific morphology when brought to 0.1 M with KCl. The similarities between Tetrahymena band C and spectrin from erythrocytes suggested further that band C might be associated with actin in Tetrahymena. Accordingly, we attempted to extract actin from isolated pellicles using standard pro-

Fig. 10. The presence of proteins in Tetrahymena

pellicles which comigrate with purified vertebrate muscle actin. The proteins of pellicles isolated by the detergent method are separated in the pair of gels on the left; those of pellicles isolated by the non-detergent method are on the right. Within each pair, the gel on the right also contains added muscle actin (arrows). Corresponding bands are seen in the left gel of each pair.

cedures. Although the present effort was not successful, we believe technical difficulties may have been responsible. This is suggested by the fact that bands were found in polyacrylamide gels of Tetrahymena pellicles prepared by two different methods which comigrate with purified vertebrate muscle actin in two gel systems. This result, together with the recent results obtained by Zeuthen & Celis [17], suggest that actin is probably present in isolated pellicles. Using immunofluorescence microscopy, these authors have shown that the cell surface of Tetrahymena contains a protein which cross-reacts with antibody to rabbit muscle actin. The protein or proteins represented by

Cytoskeletal

proteins in Tetrahymena

319

[ 19-211. In the present study, this complex was the only structure other than microtubules found in the residue of pellicles isolated using the detergent method after extraction using low salt. The predominance of band B in this fraction suggests that the attachment complex contains this protein. The apparent molecular weight of band B was determined previously in SDS polyacrylamide gels and found to be 145000 D [16]. It seems more likely that this protein is present in the circumciliary ring than in the terminal plate; the plate appears too complex to be made of one or a few proteins. Like the bacteriophage T4 baseplate [22], the basal body terminal plate is probably made of many proteins with relatively few copies each, therefore not seen at the level of resolution involved in the present study. The fact that the attachment complex remained firmly in place in membrane-containing pellicles after removal of the epiplasmic layer suggests that the circumciliary ring attaches directly to the membrane, rather than indirectly through the adjacent epiplasm. The results obtained suggest that band A (mol. wt 250000 D) is the major component of the kinetodesmal fiber. This conclusion is based on the fact that both the fibers and band A appeared insoluble in solutions of low ionic strength in one pellicle preparation, and both were soluble at low ionic strength in the other type of pellicle preparation. The different result observed in the two types of pellicle preparations is probably due to the presence of membranes in only one of these. The membranes may lead to an attenuation of solvent action which could explain the apparent insolubility of kinetodesmal fibers in this preparation. There is a previous report which suggests that the kinetodesmal fiber is composed primarily of a single polypeptide of moTetrahymena

Fig. 11. Comparison of soluble and insoluble Tetra-

hymena tubuhn and porcine brain tubulin in SDS polyacrylamide gels. Lane one (kft) is Tefrahymena cilia, lane 2 is a mixture of cilia and a fraction precipitated from the cytosol using vinblastine sulfate, and lane 3 is the vinblastine precipitate alone. Lane 4 is a mixture of the Tetrahymena vinblastine precipitate and porcine brain tubulin. Lane 5 contains brain tubulin alone.

band B in SDS gels of Tetrahymena pellicles is probably a major component of the structural complex by which basal bodies attach to the cell surface. This complex, which consists of a circumciliary ring and an enclosed basal body terminal plate (fig. 5), was first seen in negatively stained preparations of Paramecium pellicles by Hufnagel [ 181. The same structures were later seen in negatively stained preparations of

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lecular weight 21000 D [23]. As reported earlier, we were not able to repeat these results [6]. Our studies suggest that the major subunit of the kinetodesmal fiber is a much larger polypeptide. A fibrous layer on the inner surface of the cell membrane has been described in a number of cell types. Typically, this structure is found to consist of actin together with one or more high molecular weight proteins [14, 24, 251. It was suggested early that this membrane-associated layer may attach to integral membrane proteins and thereby regulate their lateral mobilities [24]. Subsequent work has provided much evidence for this idea [26-311. Most recently, Fowler & Bennett [28] have shown that dissociating spectrin from the membrane increases the rate of lateral diffusion of fluorescein isothiocyanate-labeled integral membrane proteins in fused, hemolysed human erythrocytes. In addition, Shotten et al. [31] have provided elegant experiments which strongly suggest that the meshwork of spectrin molecules limits the translational freedom of intramembranous particles in erythrocytes. One of the major characteristics of ciliated protozoa like Paramecium and Tetrahymena is their elaborate, speciesspecific patterns of surface-associated organelles [l-3]. It is only a minor extension of the above idea to suggest that the membrane-associated cytoskeletal elements studied in the present investigation of Tetrahymena may control the migrations and positioning of cortical structures in ciliates. Further studies with ciliates should be of importance in increasing our understanding of the formation and positioning of surfacerelated structures in cells generally. The authors wish to thank Ruth Jaeckel Williams for assistance with electron microscopy, Dr Gordon E. Stone for showing us the vinblastine precipitation method, and to Dr Marc Kirschner for samples of porcine brain tubulin. This research was supported by Exp Cell Res 123 (1979)

USPHS grant no. GM 25769. Dr Vaudaux was supported by Fonds Marc Birkigt (Geneve), Geigy JubiIaumstiftung (Basel) and The National Swiss Foundation for Scientific Research.

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