J. Cell Sci. 73, 279-297 (1985) 279Printed in Great Britain © The Company nf Biologists Limited 1985

BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF AN

ACTIN PROTEIN IN TETRAHYMENA PYRIFORMIS

E. JANE MITCHELL AND ARTHUR M. ZIMMERMANDepartment of /oology, University of Toronto, Toivnto, Ontario, Canada MSS IA1

5UMHARY

A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyrifonnisGL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecylsulphate (SDS)/lO% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of47 500 (47-5 Xl03iV/r). Partial proteolysis of this band with Staphylococcus aureus V-8 proteasefollowed by electrophoresis revealed a pattern of pcptides in which at least four peptides weresimilar to those observed after digestion of rabbit skeletal muscle actin. The 47-5 X 10' MT proteinappeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited byleupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column anda distinct peak was eluted with 3 M-guanidincHCl; the DNase I-binding protein appeared as adistinct band on SDS/PAGE with an apparent molecular weight of 47-5X 10\l / r . In the absenceof leupeptin, the DNase I-binding protein appeared as a broad 34x 10'\l/r band on gels. Both theATP extract and the DNase I-binding protein showed reactivity with commercially availableantiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linkedimmunosorbance assay (ELISA). Immuno-blotting- studies and affinity purification of thisantiserum showed that the recognition was not specific to the 47-5xlO3,l/r protein. However,using affinity-purified anti-actin antibodies raised against denatured actin from chick smoothmuscle, recognition of the 47-5xl03jV/r protein and a 34x 10ll/r protein was shown. In negativelystained preparations from an ATP extract after two cycles of polymerization and depolymerizationthere were filaments, 8-12nm diameter, which did not decorate with subfragment S-l of myosin,but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicatedan intensely stained 54X 103A'/r band. It is suggested that, in vitro, Tetrahymena intermediatefilaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahvmenathere is a protein that resembles actin in its extractability, molecular weight, peptide pattern afterpartial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However,this protein did not assemble into actin filaments in crude extracts.

I INTRODUCTION

Actin has been identified in numerous eukaryotic cell types. However, thepresence of actin in the ciliate Tetrahymena is controversial. Although someinvestigators have suggested or reported that actin is present in Tetrahymena(Nilsson, Ricketts & Zeuthen, 1973; Williams, Vaudaux & Skriver, 1979;Katsumaru & Fukui, 1982), others have concluded that it is not (Muncy & Wolfe,1981). Another group of investigators have reported that fibre-forming protein-38(FFP-38) may replace actin in Tetrahymena (Numata, Yasuda, Hirabayashi &Watanabe, 1980a,b) or that Tetrahymena actin has a different molecular weight and

Key words: actin, Tetrahymena, DNase I-binding protein.

280 E. J. Mitchell and A. M. Zimmerman

isoelectric point from skeletal muscle actin (Hirabayashi, Tamura, Mitsui &Watanabe, 1983). Recently, an actin complementary DNA probe has beenhybridized with messenger RNA sequences derived from division-synchronizedTetrahymena (Zimmerman, Zimmerman, Thomas & Ginzberg, 1983). Besides theevolutionary significance of the presence of actin in Tetrahymena, these cells arepotentially a useful model system for studying the role of actin in cytokinesis, since apopulation can be division-synchronized (Scherbaum & Zeuthen, 1954; Zeuthen,1971). Electron-microscopical studies of the division furrow in Tetrahymena haverevealed a microfilamentous ring (Allen, 1978; Yasuda, Numata, Ohnishi &Watanabe, 1980; Jerka-Dziadosz, 1981). A prerequisite for a study of themechanisms of assembly and functioning of the division furrow is the biochemicalidentification of the functional components.

In this paper we report our biochemical identification of Tetrahymena actin. Wesuggest that a Tetrahymena intermediate filament-like protein assembles underconditions expected to polymerize actin filaments. Part of this work has beenpresented previously (Mitchell & Zimmerman, 1982; Mitchell, Zimmerman &Forer, 1981).

MATERIALS AND METHODS

The organismTetrahymena pvrifonnis GL were maintained axenically at 28°C in medium containing 2%

(w/v) protease peptone (Difco) and 0 1 % (\v/v) liver fraction L (U.S. Biochemical Corp.)(PPL). Experimental cultures were grown aerobically. During logarithmic growth phase the cellswere harvested (104—105 cells/ml) and washed twice in non-nutrient inorganic medium aspreviously described (Bird & Zimmerman, 1980).

Preparations of pivteinsApproximately 1-2 ml of washed and packed cells (10'- 10s) were precipitated with 30-50 ml of

prechilled ( — 20°C) /j-butanol in order to remove some lipid components. After 30min at 4°C, theprecipitated material was collected by centrifugation at 12 000 £ for 30min, washed with fresh u-butanol and precipitated with 30-50 ml of prechilled (-20°C) acetone. After 1-3 h at 4°C, theacetone-precipitated material was collected by centrifugation at 12000£ for 30min and washedwith 40 ml of fresh acetone (at — 20 CC); the resulting powder (designated the acetone powder) wasallowed to dry on a glass plate at room temperature and stored at — 20 °C. Acetone powder wasextracted at about 20—40 mg/ml of HADP buffer (1 mM-A'-2-hydroxyethylpiperazine-A"-2 ethane-sulphonic acid (HEPES), p H 7 0 , 0-2mM-ATP, 1 mM-dithiothreitol (DTT), 0-01% (w/v)phenylmethylsulphonyl fluoride (PMSF)) for 30min at 4°C without further adjustment of pH. Incertain experiments 1-0/ig/ml leupeptin was also included in the HADP buffer (HADPL). Theresidual powder was removed by centrifugation at 12000^ for 30min and the supernatant waspassed through a Millipore filter of 45 f.im pore size; this supernatant was termed the ATP extract.Actin was prepared from rabbit skeletal muscle by the method of Forer (19786) or purchased fromSigma (A-2522); myosin subfragment S-l (S-l) was a gift from Dr A. Forer (York University).Protein concentrations were estimated by the method of Lowry, Rosebrough, Farr & Randall(1951).

Anti-actin antisenim and immunochemical studiesThe binding of anti-actin antibodies to antigen-coated wells was detected using enzyme-

Actinfrom Tetrahymena 281

conjugated immunological reagents by an ELISA as described by Engvall (1980). The rabbit anti-chicken skeletal muscle actin serum (anti-actin antiserum) was purchased from Miles (lots no.3121 and R826); this antiserum was pooled from rabbits immunized with soluble G-actin isolatedfrom chicken back muscle. Antigen-coated wells of microtiter plates (Becton, Dickson and Co.)were prepared according to McKearn (1980), using 1% (w/v) bovine serum albumin (BSA;Sigma, fraction V) to block sites not bound by antigen. For the assay, anti-actin antiserum wasdiluted with Tris-buffered saline, TBS (150mM-NaCl, lOniM-Tris-HCl, pH7-6) was added tothe wells, and they were incubated for 1-3 h at 37 CC. Unbound antiserum was removed by threerinses with 0-2% (w/v) BSA in TBS. Peroxidase-conjugated protein A and its substrate (2,2'-azino-di(3-ethylbenzthiazoline) sulphonic acid, ABTS) were purchased as a kit from ZymedLaboratories and were used according to instructions. Development of a dark green colour within15 min at 37°C after addition of substrate indicated a positive reaction in a well. Plates were scored

for positive reaction, either ' + ' for a dark green colour, or ' + + ' for a very dark green colour, ' 'for no reaction and ' —+ ' for a slightly positive reaction as indicated by a green colour just visiblydarker than background.

Immuno-detection of proteins separated on sodium dodecyl sulphate (SDS)/polyacrylamidegels was performed essentially according to Towbin, Staehelin & Gordon (1979). Proteins wereblotted electrophoretically from a gel to nitrocellulose paper (Schleicher & Schuell) by applying acurrent of 500 mA for 1 h using a 12 V d.c. power supply. The blotting buffer contained 25 ITIM-Tris, 200mM-glycine (pH 83) , 0 1 % (w/v) SDS, 20% (v/v) methanol (Nielsen^ al. 1982). Thenitrocellulose paper containing blotted proteins was fixed with a methanol:acetic acid:water(5:5:1, by vol.) solution for 15 min (VVeihing, 1983), before blocking unbound nitrocellulose siteswith 2% (w/v) BSA in TBS for 30min. After three rinses, each 5-10min in 0-2% (w/v) BSA inTBS, the blot was incubated with anti-actin antiserum (at a 1 :10 dilution in the rinse solution) forabout 18h at 4CC. Unbound antibodies were removed by three rinses before the blot wasincubated with peroxidase-conjugated protein A at 50/xg/ml in TBS for 30 min. The substrate,diaminobenzidine (Sigma) was freshly prepared and used according to Peferoen, Huybrechts &DeLoof (1982), after the blot was thoroughly rinsed to remove excess protein A solution. All stepswere carried out with gentle agitation at 20CC except where noted.

Affinity-purified anti-actin antibodies were prepared according to Herman & Pollard (1979)with a slight modification. G-actin (Sigma) in 0-1 m\i-CaCl2, 0 2 M - K C 1 , 0-lM-NaHCO3(pH8-5) was coupled to CNBr-activated Sepharose 6MB (Sigma). A lml sample of anti-actinantiserum was diluted to 10 ml in TBS and mixed with 1-5 ml of Sepharose 6 MB containing3-0mg actin for 20h at 4CC. The mixture was placed into a column (08cmX40cm, BioRadEcono Column) and the unadsorbed fraction was washed away with 0-01 M-Tris (pH7-6). Theadsorbed anti-actin antibodies were eluted with 0-1 M-glycineHCl (pH2-75), immediately neu-tralized by addition to 1 M-Tris (pH 7-6) and dialysed against TBS. The estimated concentrationof affinity-purified anti-actin antibodies was 8 /xg/ml, based on an extinction coefficient /•.'2x<> of 1 -4for immunoglobulin G (IgG).

Gel electrophoresisOne-dimensional SDS/polyacrylamide gel electrophoresis (SDS/PAGE) was carried out using

a microslab gel system as previously described (Bird & Zimmerman, 1980). Running gels andstacking gels were prepared as 10% (w/v) and 4% (w/v) acrylamide, respectively. The gels werejstained with 0 2 5 % (w/v) Coomassie Brilliant Blue R-250 dissolved in a methanol:aceticacid:water (50:7-5:42-5, by vol.) solution and destained with a methanol:acetic acid: water(5:7-5:87-5, by vol.) solution. Silver staining was carried out according to Wray, Boulikas, Wray& Hancock (1981). Low molecular weight standards were purchased from BioRad: 92500, 66200,45000, 31 000, 21 500, 14400jl/r. Gels were scanned using an Ortec densitometer with a greenfilter (570 nm). The baseline was drawn from the lowest point at either end of the trace, asdescribed by Low, Chaponnier & Gabbiani (1981). The peaks were cut out of the scan, weighedand expressed as a percentage of the total weight of the trace.

Peptide mappingThe procedure of Cleveland, Fischer, Kirschner & Laemmli (1977) was followed. The

282 E. jf. Mitchell and A. M. Zimmerman

47-5xl03iV/r band in the Tetrahymeiia ATP extract was excised from a preparative gel. The gelpieces containing either the Tetrahymeiia 47-5X 103

Jl/r band or actin standard were placed intowells of the peptide mapping gel, which was prepared as 15% (w/v) and 4% (w/v) acrylamide forthe running and stacking gels, respectively. Each gel piece was overlaid with 0-125-1-0/xgStaphylococcus aureus V-8 protease (sp. act. 510 units/mg, Miles) and digestion was carried outfor 30min with the power source turned off, once the tracking dye reached the bottom of thestacking gel. Fragments were separated in the running gel and detected by silver staining. Asample of S. aureus V-8 protease solubilized in the electrophoresis sample buffer was also run onpeptide mapping gels as a reference standard.

DNase I-affinity chromatographyApproximately 20ml of DNase I coupled to Sepharose-4B (Worthington) was packed into a

polypropylene column, 0-8cmX4-0cm (BioRad Econo Column), and equilibrated with severalvolumes of 5mM-CaCl2, 1 mM-HEPES (pH7-0). All procedures were performed at 4°C.Tetrahymena ATP extract containing 1-0/xg/ml leupeptin was applied to the column, which waswashed and eluted according to the method of Lazarides & Lindberg (1974) for isolation of actinsfrom non-muscle cells. The following freshly prepared solutions were applied successively to thecolumn: (1) HADPL containing 0-15.M-NaCl; (2) buffer A (30% (v/v) glycerol, lniM-CaCl2,1-0/j.g/ml leupeptin, 0-5M-sodium acetate, pH6-5); (3) buffer A containing 0-75M-guani-dine-HCl (Schwartz/Mann, ultrapure grade); (4) buffer A containing 3M-guanidine-HCl.Fractions of about 1-0 ml (20min) were collected using an I SCO model 270 fraction collector andmonitored at 280 nm. Peak fractions were pooled and dialysed against several changes ofdouble-distilled water before lyophilization and analysis by SDS/PAGE.

Preparation of filamentsAn ATP extract was centrifuged at 100000 jffor 2h at 4°C in a Beckman L3-50 ultracentrifuge.

Polymerization of protofilamentous units in the supernatant (protein concentration l-2mg/ml)was induced by the addition of KC1 and MgCl2 to concentrations of 0 1 M and 2 HIM, respectively.After this solution was gently shaken for 1 -5 h at 20 °C, KC1 was added to bring its concentration to0-6M, according to Spudich & Watt (1971). After centrifugation at 80000#for 16h at 20°C, thepellet was rinsed, then resuspended in 2 ml of HADP buffer and dialysed against two changes of 1litre of this buffer for approximately 60 h to depolymerize the filaments. The opaque solution wascentrifuged at 80000g for l h at 4°C. The resulting clear supernatant was concentratedapproximately 10-fold with Sephadex G-200 (Pharmacia), which was placed onto a dialysis saccontaining the sample. Repolymerization of the filaments was induced as before and gycerol wasadded to 25 % (v/v) to stabilize the filaments, which were stored at —20°C (final proteinconcentration about 0-2-0-4 mg/ml).

Electron microscopyRabbit skeletal muscle actin (about 0-1 mg/ml) or Tetrahymeiia filaments (0-2-0-4 mg/ml)

were applied to collodion-coated grids, rinsed with 100-200/xg/ml bacitracin and stained with 1 %(w/v) uranyl acetate in the manner described by Forer (19786). Grids were similarly preparedwith mixtures of actin/S-1 or Tetrahymeiia filaments/S-1 (S-l at a concentration of 0-8 mg/ml),which had been incubated together for 5-10 min at 20°C. Specimens were examined on a PhilipsEM201 electron microscope.

RESULTS

Electrophoresis of the Tetrahymena ATP extract and the effects of leupeptin

An acetone powder was prepared from log growth Tetrahymena and extractedwith a buffered solution containing ATP. The extract, designated the ATP extract,contained severaF proteins that were observed on SDS/polyacrylamide gels. A major

Actinfrom Tetrahymena 283

peak observed on densitometric tracings of gels migrated with an apparent molecularweight of 47-5 X 103, just behind rabbit skeletal muscle actin, which had an apparentmolecular weight of 45-5x10 in our system (Fig. 1).

In order to test for endogenous proteolytic activity, ATP extracts of Tetrahymenaacetone powder were prepared in the presence of various concentrations ofleupeptin, a known inhibitor of Tetrahymena proteases, stored for 16-18 h (at 4°C)and analysed by SDS/PAGE. The major difference between ATP extracts preparedin the presence or absence of leupeptin was in the intensity of Coomassie Bluestaining of certain protein bands. The 47-5X 103A7r band showed a markedreduction in staining intensity in ATP extracts prepared in the absence of leupeptinas compared to those extracts prepared in the presence of leupeptin (Fig. 1).

A densitometric analysis of a gel containing ATP extracts prepared in the presenceand absence of leupeptin indicated that the 47-5xlO3Mr peak accounted for 4-04,3-62 and 3-20% of the total protein in the presence of 0-1, 1-0 and 10-0/xg/mlleupeptin, respectively (Fig. 1). In the absence of leupeptin the 47-5xl03Mr peakaccounted for 2-74 % of the total protein. The peak at 54X 103A/r was also reduced inthe absence of leupeptin (1-64%) compared to ATP extracts prepared in thepresence of leupeptin (2-52, 1-70, 1-55% for 0*1, 1-0, 10-0/u.g/ml, respectively),but to a lesser extent than for the 47-5 X 103Mr peak. A third peak, at 42x 103A7r wasseen only on the densitometric profiles of SDS/PAGE of ATP extracts derived fromlate log cultures. The amount of material in this 42x 103 A/r peak was reduced to agreater extent than the 47-5 or 54xlO3A/r peaks. In the absence of leupeptin the42xlO3Mr peak represented 0-18% of the total protein and 1-60, 1-59, 1-74% at0-1, 1-0, 10-0/xg/ml leupeptin, respectively.

In the low molecular weight range, below 30xl03 the amount of protein wasreduced in the presence of leupeptin in some experimental series. The amount ofmaterial in the 26x10 Mr peak was reduced in the presence of leupeptin in two ofthree experimental series. Another peak at 29-5X 103A/r was greatly reduced in oneseries of experiments. This may be attributed to fragments of higher molecularweight proteins that were produced by endogenous proteolytic activity present inATP extracts prepared without leupeptin. The peaks below 20xl03iV/r weredifficult to distinguish; however, the pattern of peaks was variable over the range ofleupeptin concentrations studied.

Overall, there appeared to be endogenous protease activity present inTetrahymena ATP extracts, which was inhibited by leupeptin at 0-1-1-Oju.g/ml andwas not affected by 0-01 % (w/v) phenylmethylsulphonyl fluoride. It appeared thatcertain ATP extract proteins, in particular the 47-5xlO3A/r band, were moresensitive than other proteins to this endogenous proteolytic hydrolysis that wasinhibited by leupeptin. Most of the peaks also exhibited a decreasing proportion ofthe total ATP extract protein over the 0-1-10-0 pig/ml concentration range ofleupeptin. This may have been due to the relative increase in sensitive peaksprotected from proteolysis by increasing concentrations of leupeptin. Finally, theeffects of leupeptin on certain peaks, including those at 47-5, 42, 36-5 and29-5xlO3Mr appeared to be more pronounced with ATP extracts derived from late

284 E.jf. Mitchell and A. M. Zimmerman

Leupeptin (/-ig/ml)C r O i t o icT Actin Stds

54 •47-5^

36-5 •

26 *-

x10"3

- -92

-66

-45

— -21

B Control54 47-5 36-5 29-5 26

I I \ \ \

0-1 /ig/ml leupeptin

I i \ \ \

I

topFig. 1

bottom

Actin from Tetrahymena 285

Table 1. Relative proportion (%) of ATP extract peaks in the presence ofvarious concentrations of leupeptin

p , Leupeptin concentration (/ig/ml)

xp.

1231233123123123

0

1-642-541-412-743-09

<2-390-181-582-061-043-594166-816-273-842-88

0-1

2-522-951-874-043-713-541-602112-79

•693-985-194-063-344-352-74

1-0

1-702-632-033-623-244-131-592-272-44

•724-655-155-643-874-632-28

10

1-552-521-743-203-183-701-742-082-28

•434-654-774-634-024-352-09

54

47-5

42-536-5

29-5

26

Densitometric analysis of the effects of various concentrations of leupeptin on different peaksseen on scans of SDS/10% polyacrylamide gels of ATP extracts derived from Tetrahymenaacetone powder. Relative proportion was calculated by: (weight of peak/total weight oftrace)X 100. For experiments 1 and 2, acetone powders were derived from early log cultures; forexperiment 3, a late log culture. Experiment 1 is shown in Fig. 1.

log cultures as compared to those from younger cultures (Table 1, exp. 3). Thesedata are summarized in Table 1.

Peptide mapping

In order to test for similarities between the major 47-5 X 103Mr band excised froma SDS/polyacrylamide gel and skeletal muscle actin, peptide maps produced bydigestion with S. aureus V-8 protease were compared on a 15% (w/v) poly-acrylamide gel (Fig. 2). At least four distinct major fragments common to the47-5XlO3A/r Tetrahymena ATP extract band and the actin standard were detectedby silver staining. The less-prominent bands may be due to comigrant proteins in

Fig. 1. A. A SDS/10% polyacrylamide gel showing ATP extracts from an acetonepowder of Tetrahvmena prepared in the presence of various concentrations of leupeptin.Control (lane C), without leupeptin; 0-l^.g/ml leupeptin; 10/xg/ml leupeptin;10-0/xg/ml leupeptin; muscle actin, molecular weight standards (Stds). All ATP extractlanes contained 7-5 jig total protein. Bands at 54, 47-5, 36-5, 29-5, 26xlO'\Ur areindicated; muscle actin is at 45-5. B. Densitometric tracings of a SDS/10%polyacrylamide gel showing ATP extracts from Tetrahymena acetone powder preparedin the presence of various concentrations of leupeptin. Control, without leupeptin;0-1/i.g/ml leupeptin; 1-0/ig/ml leupeptin; • 100/ig/ml leupeptin. These scans werederived from the gel shown in A. Peaks at 54, 47-5, 365, 295, 26X 10'.l/r are indicatedby arrows.

286 E. J. Mitchell and A. M. Zimmerman

47-5x10 H b a n d ActinProtease'0-5 0-1251 ' 10 0 5 '

Fig. 2. Peptide maps of the 47-5x10 Mr band from Tertrahyniena and muscle actin.An ATP extract was fractionated on a SDS/10% polyacrylamide gel. The 47-5X \03Mr

band was excised from the gel. This material was digested with S. aureus V-8 protease(sp. act. 510units/mg), fractionated on a SDS/15% polyacrylamide gel and thefragments were detected by silver staining. Muscle actin was treated in a similar fashion.The 47-5xlO3Mr band was digested with 0'5/u.g and 0-125/xg protease; muscle actinwas digested with 1'0/xg and 0-5 fxg protease. Similar fragments are indicated byarrowheads.

Table 2. ELJSA of various concentrations of a Tetrahymena ATP extract ivithanti-actin antiserum

ATP extractdilution

1:01:11:21:51:101:25BlankActin

Protein(/xg/well)

54271894-92-08

_0-825

1:10

Anti-actin dilution

1:50 1:250 1:1250 1:6250 1:31250

ELISA of various concentrations of a Tetrahymena ATP extract coated onto wells of amicrotiter plate with anti-actin antiserum. ATP extract dilutions were tested against a serialdilution series of anti-actin antiserum. Positive responses were scored for ATP extract at9-54pig/well with a 1:10 anti-actin antiserum dilution. Controls included a blank, with no antigencoated onto a well, and muscle actin.

Actinfrom Tetrahymena 287

the 47-5xlO3Mr band that was excised from the preparative gel; and endogenousproteolytic breakdown of the 47-5xl03jV/r band in the ATP extract that wasprepared in the absence of leupeptin. A comparable experiment using chymotrypsininstead of 5. aureus V-8 protease indicated that after digestion of the 47-5xlO3Mr

band at least four fragments were similar to those observed after digestion of actin.

Immunochemical assay of the Tetrahymena ATP extract with anti-actin antiserum

Varying concentrations of ATP extracts from Tetrahymena acetone powder werecoated onto microtiter plates and assayed for reactivity with anti-actin antiserum byan ELISA using peroxidase-conjugated protein A and ABTS as the substrate. At a1: 10 dilution of the antiserum, positive reactions were obtained over the range of9-54/xg/well of ATP extract protein. There was no positive response by ELISAupon further dilution of the ATP extract or upon dilution of the antiserum (Table2). In control experiments with rabbit skeletal muscle actin, a minimum of0-08/ig/well of actin was necessary to record a positive ELISA reaction withdilutions of the antiserum ranging from 1:10 to 1:1250.

In order to test the specificity of the positive ELISA reaction with anti-actinantiserum and Tetrahymena proteins, immuno-blotting experiments were per-formed. An ATP extract was fractionated on a SDS/polyacrylamide gel and electro-blotted onto nitrocellulose. A colorimetric method was used to detect antibodiesbound to protein on the nitrocellulose (Peferoen et al. 1982). At a 1:10 dilution ofantiserum, several ATP extract bands were detected, including about eight bandsbelow 35xl03A/r and two bands in the range of about 46-52(XlO3)iV/r. Hence, thereactivity between the Tetrahymena material and the anti-actin antiserum was notspecific to the 47-5xlO3Mr band. In control experiments, the blotted ATP extractproteins were incubated with peroxidase-conjugated protein A followed by substratewithout initial treatment with antibody. No bands-were detected, although some lowmolecular weight bands that appeared to correspond to those detected by theantibody remained white against the light pink background. Thus, the bands below35xlO3Mr that were detected reflect a non-specific reactivity of the antiserum. Inorder to analyse further the reactivity of the two 46-52(x 103)Mr bands, affinity-purified anti-actin antibodies were prepared by preadsorption of the antiserum torabbit skeletal muscle G-actin coupled to Sepharose. No Tetrahymena bands weredetected after affinity-purified anti-actin antibodies were incubated with blottedTetrahymena proteins. The skeletal muscle actin standard was detected, indicatingthat the affinity-purified antibodies had not lost their reactivity during the pread-sorption procedures. Hence the anti-actin antiserum did not specifically react withTetrahymena actin. This may be attributable to the fact that native G-actin in thesoluble form was used to immunize the rabbits rather than denatured actin.

Another series of immuno-blotting experiments were conducted using affinity-purified anti-actin antibodies, a gift from Dr Jane Aubin. This material wasprepared in rabbits immunized with actin derived from smooth muscle of chick. Theactin antigen was purified by SDS/PAGE. The 47-5 and 34Xl03iV/r protein bands

288 E. J. Mitchell and A. M. Zimmerman

2-0

1-5

1-0

!<

0-5

>2-0 Buffer A

u IUII I h

IV

GuanidineHQ

I 0-75 M I 3M

v vi

15 30 45Fraction number

60 75

Fig. 3. Elution profile for DNase I-Sepharose affinity chromatography of an ATPextract, from Tetrahymena acetone powder, showing absorbance readings at 280 nm foreach fraction. The column was washed with HADPL containing 015 M-NaCl and waseluted stepwise as indicated with buffer A (30% (v/v) glycerol, 1 m:\1-CaCl2, 1-Oju.g/mlleupeptin, 0-5 M-sodium acetate, pH6-5); buffer A containing 0'75 M-guanidine-HCl;buffer A containing 3 M-guanidine-HCl. Fractions were pooled, as indicated by i, ii, iii,iv, v, vi, and prepared for analysis by SDS/polyacrylamide gels.

present- in Tetrahymena ATP extracts were detected in immuno-blotting experi-ments. In addition, some low molecular weight bands were detected; these corre-sponded to the white bands seen in control experiments with peroxidasereagent and substrate, in the absence of antibody. Thus, the anti-actin antibodiesraised against SDS-denatured actin recognized the 47-5 X 10" Mr TetraJiyinena actin.Furthermore, the observed reactivity with the 34X 10' Mr band was consistent withthe possibility that this protein was a proteolytic breakdown product of actin.

DNase I-affinity cluvmatography

An ATP extract (about 6-0 mg total protein) of Tetrahymena acetone powderprepared with 1-0/ig/ml leupeptin was applied to a DNase I column. A single peakof DNase I-binding material was eluted with 3 M-guanidine-HCl (Fig. 3). Analysis

Actin from Tetrahymena 289

D . . . .. GuanidineHCIPooled fractions I 0 . 7 5 M 3 M 1

Stds Actin i ii iii iv v vi Actin

X10"3

92-

66-

45-

31-'

Fig. 4. The pooled fractions of Tetrahymena ATP extract eluted from a DNaseI-Sepharose column were analysed by a SDS/10% polyacrylamide gel stained withCoomassie Blue. The various fractions shown in Fig. 3 were pooled and prepared forelectrophoresis. For the material in pooled fractions i—iv only pooled fraction i containedmaterial detectable by Coomassie Blue. Fraction v contained material eluted by buffer Acontaining 0-75 M-guanidine'HCl. Fraction vi contained the DNase I-binding materialeluted by buffer A containing 3 M-guanidine-HCl. A 47-5X 103A/r band (arrowhead) ispresent in the DNase I-binding material; this band represents about 20% of the DNaseI-binding material excluding polypeptides below 25Xl03il/r. Muscle actin andmolecular weight standards (Stds) are also shown.

of this peak by SDS/PAGE confirmed the presence of a 47-5 X 103Mr protein band,Tetrahymena actin (Fig. 4). Densitometric analysis indicated that this protein bandwas about 20% of the protein material eluted with 3 M-guanidine-HCl, excludingthe low molecular weight material (below 25xl03A/r). This peak material waspositive when assayed by ELISA with anti-actin antiserum. However, the47-5xlO3Mr protein did not react with the affinity-purified antibodies in immuno-blotting experiments. The reactivity of the DNase I-binding material with anti-actinantibodies produced against SDS-denatured actin was not determined. In theabsence of leupeptin, the DNase I-binding material from [35S]methionine-labelledTetrahymena migrated on a SDS/polyacrylamide gel as a broad band of 34x 103 A/r;this material may have been a proteolytic cleavage product of Tetrahymena actin.

290 E. jf. Mitchell and A. M. Zimmerman

Tetrahymena filaments

Filaments with an apparent width of 8-12 nm were prepared from an ATP extractof Tetrahymena acetone powder by two cycles of polymerization and depoly-merization. These filaments had a different appearance from actin filaments; their

^ 6

8Fig. 5. Electron micrograph of negatively stained rabbit skeletal muscle actin filaments.The filament has a diameter of 7-9 nm. X 139000.

Fig. 6. Electron micrograph of negatively stained Tetrahymena filaments prepared byin vitro polymerization of an ATP extract derived from a Tetrahymena acetone powder.The filament has a diameter of 8-12nm. X 139000.

Fig. 7. Rabbit skeletal muscle actin filaments decorated in vitm with myosin subfrag-ment S-l showing 'arrowhead' morphology. X 150000.

Fig. 8. High-power electron micrograph of negatively stained Tetrahymena filamentsillustrating the thin strands (some of which are indicated by arrows) that comprise thedouble strands evident at low magnification. X214000.

Actin from Tetrahymena 291

diameter was larger and they exhibited a 'double-strandedness' (Figs 5,6). We couldnot establish unequivocally the myosin subfragment 1 (S-1) decoration of theseTetrahymena filaments although muscle actin decorated with S-1 showed the typicalarrowhead morphology (Fig. 7). Careful examination of the Tetrahymena filamentsat higher magnification suggested that these filaments were actually composed offour or more thin strands, approximately 3 nm in width, rather than two strands(Fig. 8). Each of the two strands seen at low magnification appeared to be composedof two of these thin strands and the double strands were intertwined with oneanother at regular intervals. This arrangement possibly gives rise to the double-strandedness consistently observed at low magnification.

Examination of a sample of the Tetrahymena filaments by SDS/polyacrylamidegels indicated an intensely stained band at 54xl03iV/r, a broad band at 34xlO3Mr

and some weakly stained bands, including one at 47-5X 103Mr with silver stain (Fig.9).

cCD

E E

< iZx10"3

9 2 -66-

f~ -<5445-

>3431-

21-

Fig. 9. A SDS/10% polyacrylamide gel of a sample of Tetrahymena filament materialprepared by in vitro polymerization of an ATP extract derived from a Tetrahymenaacetone powder (as in Figs 6, 8). The major band with silver staining is S4xlO3.l/r, abroad 34 band and a 47-5xl03jUr are also indicated. Muscle actin is shown forcomparison.

292 E. J. Mitchell and A. M. Zimmerman

DISCUSSION

In general these studies show that Tetrahymena contain actin. This was identifiedusing several criteria. The protein was identified biochemically by comigration onSDS/polyacrylamide gels, peptide mapping, DNase I-affinity chromatography andreactivity with anti-actin antibodies. Although structural analysis of negativelystained material, prepared from two cycles of polymerization and depolymerization,confirmed the presence of filaments, these differed from normal actin filaments intheir appearance and in their lack of reactivity with subfragment S-l of myosin.

Identification of actin in an ATP extract of Tetrahymena acetone powder

The starting material used in all the procedures reported here was an acetonepowder of Tetrahymena that was extracted with buffer containing ATP, whichsolubilizes G-actin from acetone powders of many muscle and non-muscle cells (cf.reviews by Pollard & Weihing, 1974; Forer, 19786). Initially, it was thought thatacetone precipitation of Tetrahymena cells at — 20 °C would cause denaturation ofany endogenous proteolytic enzymes. This supposition was not true since our resultsshowed a proteolytic breakdown of actin in the chilled acetone precipitation of cells.Both Williams et al. (1979) and Numata et al. (1980fl,6) performed an acetoneprecipitation of Tetrahymena and were not able to identify actin. In another attemptto demonstrate Tetrahymena actin, Hirabayashi et al. (1983) reported that theprotease activity was high in whole-cell extracts and it was necessary to use8M-guanidine-HCl to prevent protein degradation. However, they could notidentify a protein having the same molecular weight or isoelectric point as muscleactin. Recently, Banno, Yano & Nozavva (1982) showed that extracellular andintracellular proteases from Tetrahymena were inhibited by about 90 % with1 /xg/ml leupeptin. Leupeptin is a peptide (acetyl-i.-leucyl-i.-leucyl-i.-arginal)(Umezawa, 1972), which was shown to inhibit thiol-requiring proteases ofTetrahymena (Levy, Sisskin & McConskey, 1976). In the present study, the majordifference seen on SDS/polyacrylamide gels for ATP extracts of Tetrahymenaprepared with and without leupeptin was in the staining intensities of certain bands.This result suggested the presence of a protease(s) that catalysed the hydrolysis ofspecific polypeptides in Tetrahymena, in particular actin. Our results demonstratedthe importance of using inhibitors such as leupeptin during subcellularfractionations of Tetrahvmena. It was possible that Tetrahymena actin washydrolysed specifically by endogenous proteases, and this may be an explanation forthe previous unsuccessful attempts to isolate Tetrahymena actin. The broad34xlO3Mr band seen on SDS/polyacrylamide gels of both the DNase I-bindingprotein in the absence of leupeptin and the filament preparation is consistent withthe size of a protease-resistant G-actin fragment reported by others (Adelman, 1977;Jacobson & Rosenbusch, 1976; Mantulenko, Khaitlina & Shelud'ko, 1983).Morever, both 47-5 and 34x10Mr bands reacted with anti-actin antibodiesproduced against denatured actin in immuno-blotting experiments.

Actinfrom Tetrahymena 293

The ATP extract of Tetrahymena acetone powder showed reactivity by anELISA with anti-actin antiserum that was raised against native actin from chickenskeletal muscle. This suggested recognition of a determinant of muscle actin incommon with a component in the ATP extract. However, upon further studies itwas found that the reactivity of the anti-actin serum was not specific for the47-5xlO3Mr Tetrahymena protein. This was shown by immuno-blottingexperiments with whole anti-actin antiserum and further by affinity purification ofthe anti-actin antibodies (cf. Forer, 1978a). On the other hand, recognition of theTetrahymena 47-5 X 103Mr actin protein as well as a 34X 103Mr protein was observedin immuno-blotting experiments with affinity-purified anti-actin antibodiesproduced in rabbits immunized with denatured actin from chick smooth muscle.The 34X 103Mr protein may be a proteolytic breakdown product of actin. A possibleexplanation for the lack of recognition of Tetrahymena actin by the commerciallyavailable antiserum involves the fact that native, soluble G-actin was used toimmunize the rabbits. It has recently been argued that anti-actin antibodies againstnative globular actin differ from those obtained by immunization with denatured ormodified actin (DeCouet, 1983). It is deemed that antibodies against a denaturedactin would be non-species-specific, since denaturation (e.g. by SDS) would resultin antibodies directed against internal determinants that are genetically conserved.In contrast, antibodies raised against native actin such as those used in the presentstudy, would tend to be directed against hydrophilic surface determinants, whichare genetically more variable and hence tend to be species-specific. This argumentwas supported in a qualitative sense in an earlier report by Groschel-Stewart et al.(1977). Furthermore, Bulinski, Kumar, Titani & Hauschka (1983) have preparedan isozyme-specific antibody by immunization with the amino-terminal trypticpeptide of actin, which is the longest variable sequence among the different types ofactins. Thus the commercially available antibodies used in the present study may betype(isozyme)-specific rather than species-specific, since the antibodies that wereraised against chicken skeletal muscle G-actin strongly reacted against rabbit skeletalmuscle G-actin. Hence, actin from Tetrahymena, presumably a non-muscle type,was not recognized by this antiserum, although it was recognized by other antibodiesproduced against a denatured smooth muscle actin.

Some degree of homology in the primary sequence of muscle and Tetrahymenaactin was suggested by the similarities in the pattern of peptides produced afterpartial proteolysis, of muscle actin and the 47-5 X 103A/r band of Tetrahymena ATPextract, by S. aureus V-8 protease and chymotrypsin.

DNase I-binding protein of Tetrahymena

The DNase I-binding protein from Tetrahymena had an apparent molecularweight of 47-5XlO3Mr as determined by SDS/PAGE. In Tetrahymena, leupeptinwas necessary for the recovery of the 47-5xlO3.l/r actin band from a DNase Icolumn. Tetrahymena actin differs from the 48xlO3.Ur actin from Entamoebahistolytica, which was reported not to bind DNase I (Meza, Sabanero, Cazares &

294 E. J. Mitchell and A. M. Zimmerman

Bryan, 1983; Gadasi, 1982). It is possible that Tetrahymena actin has a low affinityfor DNase I, as was reported for Naegleria actin by Sussman, Lai & Fulton (1980).

Filaments derived from Tetrahymena

The filaments prepared by polymerization and depolymerization of an ATPextract of Tetrahymena acetone powder were larger than actin filaments, had adouble-stranded appearance and we could not confirm decoration by subfragmentS-l of myosin. The overall size and appearance of these filaments resembled inter-mediate filaments (Steinert, Zimmerman, Starger & Goldman, 1978, their fig. IB),and the double-strandedness, in particular, was similar to the insoluble filamentouscomponents derived from flagellar axonemes of sea-urchin sperm (Linck &Langevin, 1982). Our polyacrylamide gel analysis of the Tetrahymena filamentsindicated an intensely stained band at 54 X 103jV/r, which may be consistent with anintermediate-filament class protein. A 14 nm filament from a 49xlO3Mr protein(which was formerly designated FFP-38 by Numata et al. 1980a) derived fromTetrahymena has been identified by Numata & Watanabe (1982). This materialresembled mammalian intermediate filaments with respect to molecular weight andamino-acid composition.

The specific conditions required to polymerize Tetrahymena actin in vitro havenot been identified. The results of Katsumaru & Fukui (1982) indicated that actinfilaments are present in situ. Proteolytic breakdown of actin may have preventedeffective polymerization of actin (Adelman, 1977; Jacobson & Rosenbush, 1976;Mantulenko et al. 1983). However, the pattern of bands was similar on SDS/polyacrylamide gels for these filaments prepared in the absence or presence of1 fAg/m\ leupeptin. The presence of an actin-binding protein (for reviews see Craig& Pollard, 1982; Weeds, 1982), might also account for the lack of polymerizability ofG-actin in crude Tetrahymena extracts.

We have reported biochemical evidence for the presence of actin in Tetrahymena,having an apparent molecular weight of 47-5X103. The expression at the level oftranscription of at least one Tetrahymena actin gene has been reported byZimmerman et al. (1983). In this study, poly(A)+ mRNA from specific cell-cyclestages of synchronized Tetrahymena was hybridized to actin cDNA probes andtranslated in a cell-free reticulocyte system containing [35S]methionine. Takentogether, our evidence shows that Tetrahymena contain actin. Further investigationof the factors involved in controlling Tetrahymena actin filament assembly is inprogress.

We thank Dr A. Forer for many hours of assistance and guidance with the electron-microscopical studies and for many useful discussions throughout the course of this study, Dr B.Satir for suggesting the use of leupeptin in our experiments, Dr J. Aubin for the kind gift ofaffinity purified anti-actin antibodies, MrsG. Allen for excellent typing of this manuscript and MsH. Fisher for expert artwork and techriical assistance.

This work was supported by NSERC. E.J.M. was a recipient of a NSERC postgraduatescholarship

Actinfmm Tetrahymena 295

REFERENCES

ADELMAN, M. R. (1977). Physarum actin. Observations on its presence, stability, and assembly inplasmodial extracts and development of an improved purification procedure. Biochemistry 16,4862-4871.

ALLEN, R. D. (1978). Membranes of ciliates: ultrastructure, biochemistry and fusion. CellSurface Reviews, vol. 5, Membrane Fusion (ed. G. Poste & G. L. Nicholson), pp. 657-763.New York: Elsevier/North-Holland.

BANNO, Y., YANO, K. & NOZAWA, Y. (1982). Biochemical characterization of secreted proteasesduring growth in Tetrahymena pvrifonnis WH—14: Comparison of extracellular withintracellular proteases. J. Pmtozool. 29, 91-98.

BIRD, R. C. & ZIMMERMAN, A. M. (1980). Induction of tubulin synthesis during ciliaregeneration in growing Tetrahvmena. Expl Cell Res. 128, 199-205.

BULINSKI, J. C , KUMAR, S., T I T A N I , K. & HALSCIIKA, S. D. (1983). Peptide antibody specificfor the amino terminus of skeletal muscle cv-actin. Proc. natn. Acad. Sci. U.SA. 80, 1506-1510.

CLEVELAND, D. W., FISCHER, S. G., KIRSCIINKR, M. W. & LAEM.MLI, U. K. (1977). Peptide

mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.jf. biol. Chem, 252, 1102-1106.

CRAIC, S. W. & POLLARD, T. D. (1982). Actin-binding proteins. Trends Biochem. Sci. 7, 88-92.DECOUET, H. G. (1983). Studies on the antigenic sites of actin: a comparative study of the

immunogenic crossreactivity of invertebrate actins. J. Muscle Res. Cell Motil. 4, 405-427.ENCVALL, E. (1980). Enzyme immunoassay ELISA and EMIT. Methods in Knzymology, vol. 70

(ed. H. Van Vunakis & J. J. Langone), pp. 419-437. New York: Academic Press.FORER, A. (1978a). Chromosome movements during cell-division: possible involvement of actin

filaments. Nuclear Division in the Fungi (ed. J. B. Heath), pp. 21-88. New York: AcademicPress.

FORER, A. (19786). Electron microscopy of actin. Principles and Techniques of ElectronMicroscopy, Biological Applications, vol. 9 (ed. M. A. Hayat), pp. 126-174. New York: VanNostrand Reinholt.

GADASI, H. (1982). Isolated Entamoeba histolytica actin does not inhibit DNase 1 activity.Biochem. biophys. Res. Commun. 104, 158-164.

GROSCIIEL-STEWART, U., CELRREMANS, S., LKIIR, I., MAIII.MKISTER, C. & PAAR, E. (1977).

Production of specific antibodies to contractile proteins, and their use in immunofluorescencemicroscopy. Histochemistry 50, 271-279.

HERMAN, I. M. & POLLARD, T. D. (1979). Comparison of purified anti-actin andfluorescent-heavy meromyosin staining patterns in dividing cells. J. Cell Biol. 80, 509-520.

HIRABAYASIII, T., TAMURA, R., MITSUI , I. & WATANAHE, Y. (1983). Investigation of actin inTetrahymena cells. A comparison with skeletal muscle actin by a devised two-dimensional gelelectrophoresis method. J. Biochem. 93, 461-468.

JACOBSON, G. R. & ROSENBUSCII, J. P. (1976). ATP binding to a protease-resistant core of actin.Proc. natn. Acad. Sci. U.SA. 73, 2742-2746.

JERKA-DZIADOSZ, M. (1981). Cytoskeleton-related structures in Tetrahymena themtophila:microfilaments at the apical and division furrow rings. Jf. Cell Sci. 51, 241-253.

KATSUMARU, H. & FUKUI , Y. (1982). In vivo identification of Tetrahymena actin probed byDMSO induction of nuclear bundles. Expl Cell Res. 137, 353-363.

LAZARIDES, E. & LINDBF.RG, U. (1974). Actin is the naturally occurring inhibitor ofdeoxyribonuclease I. Proc. natn. Acad. Sci. U.SA. 71, 4742-4746.

LEVY, M. R., SISSKIN, E. E. & MCCONKEY, C. L. (1976). A protease that increases during aperiod of enzymic and metabolic adjustment in Tetrahvmena. Archs Biochem. Biophvs. 172,634-647.

LINCK, R. W. & LANCEVIN, G. L. (1982). Structure and chemical composition of insolublefilamentous components of sperm flagellar microtubules. jf. Cell Sci. 58, 1-22.

Low, R. B., CIIAPONNIER, C. & GARKIANI, G. (1981). Organization of actin in epithelial cellsduring regenerative and neoplastic conditions. Correlation of morphologic, immunofluorescent,and biochemical findings. Lab. Invest. 44, 359-367.

296 E. J. Mitchell and A. M. Zimmerman

LOWRY, 0 . H., ROSEBROUGII, N. J., FARK, A. L. & RANDALL, R. J. (1951). Protein

measurement with the Folin phenol reagent. J . biol. Client. 193, 265-275.MANTULENKO, V. B., KIIAITLINA, S. Y. & SIIKLL'D'KO, N. S. (1983). A high-molecular weight

proteolysis-resistant actin fragment. Biokhimiya (Biochemistiy) 48, 61-66.MCKEARN, T. J. (1980). Binding hybridoma antibodies to polyvinylchloride microtiter dishes.

Monoclonal Antibodies (ed. R. H. Kennett, T. J. McKearn & K. B. Bechtol), pp. 388-390.New York: Plenum Press.

MEZA, I., SABANERO, M., CAZARKS, F. & BRYAN, J. (1983). Isolation and characterization ofactin from Entamoeba histolytica. jf. biol. Client. 258, 3936-3941.

MITCHELL, E. J. & ZIMMERMAN, A. M. (1982). Characterization of the actin-like protein fromTetrahymena.J. Cell Biol. 95, 281a.

MITCHELL, E. J., ZIMMERMAN, A. M. & FORKR, A. (1981). Identification of a protein presumedto be actin from Tetrahymena.J. Cell Biol. 91, 308a.

MUNCY, L. F. & WOLFE, J. S. (1981). Evidence for a non-actin inhibitor of deoxyribonuclease I(DNase I) in Tetrahymena thennophila. J. Cell Biol. 91, 303a.

NIELSEN, P. J., MANCHESTER, K. L., TOWUIN, H., GORDON, J. & THOMAS, G. (1982). The

phosphorylation of ribosomal protein S6 in rat tissues following cyclohexamide injection, indiabetes and after denervation of diaphragm. J. biol. Client. 246, 12316-12321.

NILSSON, J. R., RICKETTS, T. R. & ZKLTIIKN, E. (1973). Effects of cytochalasin B on celldivision and vacuole formation in Tetrahymena pyrifonnis GL. Expl Cell Res. 79, 456-459.

NUMATA, 0 . & WATANABE, Y. (1982). In vitro assembly and disassembly of 14-nm filament fromTetrahymena pvrifonnis. The protein component of 14-nm filament is a 49,000-dalton protein.J. Biochem. 91," 1563-1573.

NUMATA, 0 . , YASUDA, T. , HIRABAYASIII, T. & WATANAHK, Y. (1980«). Isolation and someproperties of a new fiber-forming protein from Tetrahvniena pvrifonnis. jf. Biochem. 88,1487-1498.

NUMATA, O., YASUDA, T., HIRABAVASHI, T. & WATANAHI-:, Y. (19806). A new fibre-forming

protein from Tetrahymena pyrifonnis. Expl Cell Res. 129, 223-230.PEFEROEN, M., HUYBRECHTS, R. & DELOOK, A. (1982). Vacuum-blotting: A new simple and

efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose.FEBS Lett. 145, 369-372.

POLLARD, T. D. & WEIIUNC, R. R. (1974). Actin and myosin and cell movement. Crit. Rev.Biochem. 2, 1-65.

SCIIERBAUM, O. H. & ZEUTIIEN, E. (1954). Induction of synchronous cell division in masscultures of Tetrahymena pvrifonnis. Expl Cell Res. 6, 221-227.

SPUDICH, J. A. & WATT, S. (1971). The regulation of rabbit skeletal muscle contraction. 1.Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and theproteolytic fragments of myosin. J. biol. Chem. 246, 4866-4871.

STEINERT, P. M., ZIMMERMAN, S. B., STARCI-R, J. M. & GOLDMAN, R. D. (1978).

Ten-nanometer filaments of hamster BHK-21 cells and epidermal keratin filaments have similarstructures. Proc. natn. Acad. Sci. USA. 75, 6098-6101.

SUSSMAN, D. J., LAI , E. Y. & FULTON, C. (1980). Unusual features of .Xaegleria actin. J. CellBiol. 87, 223a.

TOWBIN, H., STAEIIELIN, T. & GORDON, J. (1979). Electrophoretic transfer of proteins frompolyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn. Acad.Sci. U.SA. 76, 4350-4354.

UMEZAWA, H. (1972). Enzvme Inhibitors ofMicivbial Origin, pp. 15-29. Tokyo: University ParkPress.

WEEDS, A. (1982). Actin-binding proteins - regulators of cell architecture and motility. Xature,Land. 296, 811-816.

WEIIIING, R. R. (1983). Purification of a HeLa cell high molecular weight actin binding proteinand its identification in HeLa cell plasma membrane ghosts and intact HeLa cells. Biochemistiy22, 1839-1847.

WILLIAMS, N. E., VAUDALX, P. E. & SKRIVKK, L. (1979). Cytoskeletal proteins of the cellsurface in Tetrahvniena. I. Identification and localization of major proteins. Expl Cell Res. 123,311-320.

Actin from Tetrahymena 297

WRAY, W., BOULIKAS, T., WRAV, V. P. & HANCOCK, R. (1981). Silver staining of proteins inpolyacrylamide gels. Analyt. Biochem. 118, 197-203.

YASUDA, T., NUMATA, O., OIINISMI, K. & W ATA N A in:, Y. (1980). A contractile ring and corticalchanges found in the dividing Tetrahymena pyrifonnis. Hxpl Cell Res. 128, 407-417.

ZEUTHEN, E. (1971). Synchrony in Tetrahvmena by heat shocks spaced a normal cell generationapart. Expl Cell Res. 68, 49-60.

ZIMMERMAN, A. M., ZIMMERMAN, S., THOMAS, J. & GIN/.HLRG, I. (1983). Control of tubulinand actin gene expression in Tetrahvmena pvrifonnis during the cell cycle. FHBS Lett. 164,318-322.

(Received 30 April 1984 - Accepted 21 August 1984)