J. Cell Sci. 60, 169-179 (1983)
169
Printed in Great Britain © The Company of Biologists Limited 1983
EVIDENCE FOR THE LACK OF ACTIN
INVOLVEMENT IN MITOSIS AND IN THE
CONTRACTILE PROCESS IN SPIROSTOMUM
TERES
VALERIE S. HOBBS, ROBERT A. JENKINS
University of Wyoming, Laramie, Wyoming 82070, U.SA.
AND JAMES R. BAMBURG
Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523,
U.SA.
SUMMARY
Studies using actin decoration techniques and electron microscopy have failed to show the
presence of actin in the ciliate Spirostomum teres. The internal and external membranes of dividing
cells were made permeable by treatment with Triton. The myosin subfragment S-l was introduced
into the cells for incubation under conditions suitable for actin decoration. Arrowhead decoration
of microfilaments was not observed within dividing micronuclei or in cytoplasmic filament bundles.
However, using similar procedures, the brush border of mouse small intestine yielded clearly
decorated microfilaments. A spectrophotometric DNase I inhibition assay specific for G-actin
demonstrated that the level of actin in 5. teres extracts was less than 0-06% of the total soluble
protein and confirms the observations made using the decoration procedures. On the basis of this
work, it appears that actin does not play a significant part in either mitotic chromosome movement
or contractility in 5. teres.
INTRODUCTION
Since Huxley (1957) first identified actin as a contractile protein in muscle tissue
it has been found and associated with motility in numerous non-muscle cells (for
reviews see Forer, \978a,b; Korn, 1978). The universality of actin has led to the
generalization that actin may be assumed to be involved in all cellular motile systems
until proven otherwise (Forer, 1978a). However, the question of actin's role as a force
producer for chromosome movement has still to be answered. Spirostomum teres, a
large and highly contractile heterotrich ciliate protozoon, seems to have advantages
as a model for determining the presence and role of actin in the division apparatus.
These cells have a large oval macronucleus that divides by an amitotic process,
together with from three to five micronuclei that go through mitosis synchronously.
The micronuclear envelope remains intact (intranuclear mitosis), and the mitotic
spindle displays essentially parallel microtubules at all post-metaphase stages.
Subfragment S-l labelling of actin followed by electron microscopy was chosen as
the preferred procedure for locating actin. The S-l subfragment of the myosin
molecule has the advantage of binding to only one actin site, thus precluding the crosslinking of actin filaments observed with heavy meromyosin (HMM) (Trinick & Offer,
1979). This technique was supplemented with a biochemical one, the DNase inhibition assay (Blikstad et al. 1978; Harris, Bamburg, Bernstein & Weeds, 1982).
12
CEL 60
170
V. S. Hobbs, R. A. Jenkins andjf. R. Bamburg
In this paper data are presented indicating that this ciliate does not employ actin
in chromosome movement or in its cytoplasmic contractile process. We suggest that
contractility in S. teres involves a calcium-dependent, non-actin, filamentous system
similar to that described for the vorticellid spasmoneme (Routledge, 1978).
MATERIALS
AND
METHODS
The organism
S. teres were cultured in a Cerophyl-lettuce medium with Pseudomonas ovalis and Bacillus
subtilis as bacterial food. Synchronization of division in mass cultures was achieved by initiating
cultures from stationary-phase organisms; a high proportion of these cells divided approximately
36 h after inoculation. Organisms in known stages of division were selected for the ultrastructural
studies. Asynchronous cell populations were used for the DNase I inhibition assay.
Preparation of proteins
Rabbit skeletal muscle myosin was prepared by a modification of the method of Kielley &
Harrington (1959). The S-l subfragment was prepared by digestion of this myosin with papain,
according to the method of Margossian & Lowey (1973). The pellet from myosin extraction was
saved and muscle acetone powder was prepared after extraction with water (Forer, 1978a). The
acetone powder was dried and stored at —20 °C. Actin was prepared from this powder by the method
of Spudich & Watt (1971). Protein concentrations were assayed by Bio-Rad dye binding assay (BioRad Laboratories, Richmond, Calif.) and by the method of Lowry, Rosebrough, Farr & Randall
(1951). The ATPase activity of both myosin and S-l was determined by the method of Pollard &
Korn (1973). Bovine pancreatic DNase I (Sigma Chem. Co., St Louis, Mo.; Type DN-EP) was
stored at - 2 0 c C in small volumes as a concentrated stock solution (5-10mg/ml) in 125 mMTrisHCl (pH7-5), 5 mM-MgCl2 , 2mM-CaCl 2 , 1 niM-NaN3 and diluted to 50 Jig/ml in this buffer
24 h before use.
Gel electrophoresis
Polyacrylamide gels ( 7 5 % ) were prepared by the general procedure of Shapiro, Vinuela &
Maizel (1967). Gels contained 0 1 % sodium dodecyl sulphate (SDS) and 2-5% Tris-glycine
buffer. Gels were stained in 0-25% Coomassie Blue in 50% methanol/10% acetic acid and destained overnight in 7-5% acetic acid/5% methanol. Samples of actin, myosin and S-l were
electrophoresed simultaneously.
• Permeabilization of cells to S-l
Selected cells were washed in balanced salt solution and pre-fixed in 0'05—O'l % glutaraldehyde
in 40 mM-sodium phosphate buffer to help preserve fine structure. This concentration is considered
too weak to inhibit S-l penetration (Ohtsuki, Manzi, Palade & Jamieson, 1978). The cells were then
placed in buffered 001 % Triton X-100 (30mM-KCl, lOmM-EDTA, 5mM-MgSO 4 , 10mM-Trismaleate, 0-2% 2-mercaptoethanol, pH7-3) for from 1-15 min. Following exposure to buffered
detergent, cells were washed in buffered saline and placed in buffered S-l (final S-l concentration,
~ 2 mg/ml). Cells were incubated in the S-l solution at 10°C for 30 min, then at room temperature
for 60-120 min. Controls were incubated in buffer only, or in an S-l/ATP mixture containing
20mM-ATP (Sigma A5394 vanadium-free, crystalline adenosine 5'-triphosphate-3H2O). Incubated cells were rinsed in buffer or, in the case of ATP controls in buffer plus 20mM-ATP, and
fixed for electron microscopy.
Electron microscopy
Fixation for electron microscopy was at room temperature with 5 % (v/v) glutaraldehyde in
40 mM-phosphate buffer (pH7-2) for 15 min followed by 1% (w/v) OsO* (pH6-8) in the same
buffer for 1 h. In some instances 0-05—0-2 % tannic acid was added as a powder to the fixative (Begg,
Lack of actin in Spirostomum teres
171
Rodewald & Rebhun, 1978) to enhance the visibility of the arrowhead configuration; in these cases
only sodium phosphate buffers were used, to prevent formation of a potassium-tannic acid
precipitate. Cells were washed thoroughly with buffer before post-fixation in OsO 4 . Cells were
processed for electron microscopy by usual procedures and sectioned with glass knives.
S-1 decoration of actin
Negative staining of actin filaments. Rabbit skeletal muscle actin (0-25mg/ml in SOmM-KCI,
2mM-MgCl2 , 1 mM-Tris'HCl, pH 8) was applied to Formvar/carbon-coated grids and negatively
stained with 2 % uranyl acetate. Grids were similarly prepared with an actin/S-1 (S-1, 2mg/ml)
solution, which had been incubated at room temperature for 5 min, then rinsed after 1 min with
Bacitracin (0*01 %) followed by uranyl acetate.
5-7 treatment of rat intestinal brush borders. As a further confirmation of the binding ability of
the S-1 used in experiments, S-1 was used to decorate actin filaments in the intestinal brush border
of 12-day-old Sprague Dawley rats. Brush borders were demembranated by the method of Mooseker
& Tilney (1975) and incubated with S-1 (2mg/ml) for 3-5 h at 4°C and 0-5 h at room temperature.
The pellets of isolated demembranated brush borders were then fixed in glutaraldehyde and OsO.»,
embedded, and sectioned for electron microscopy.
Extracted cell models
Tritonized contractile models of S. teres were prepared as previously described (Jenkins, 1974)
and exposed to Ca 2 + /EGTA buffer solutions without ATP.
DNase I assay
Cultured 5. teres were washed in balanced salt solution and pelleted by low-speed centrifugation.
The cell pellet of approximately 0 4 ml was combined with actin extraction buffer (2mM-Tris-HCl,
0-2mM-ATP, 0-5mM-dithiothreitol, pH8) to a final volume of 1 ml. To 0-25 ml of this suspension
50 fii of F-actin (0-1 mg/ml) was added to be used as a control; this mixture and the other 0-75 ml
were sonicated separately twice (5 s each), cooled in an ice bath for 5 min, then centrifuged in a
Beckman Airfuge for 15 min at 4°C at 170000g. The supernatants containing the extract from 5.
teres were used for the DNase I inhibition assay, protein assay, and gel electrophoresis. The pellets
were completely solubilized by sonication in 200 fA of the extraction buffer containing 1 % Triton
and samples were also used for the DNase I inhibition assay. The DNase I inhibition assay was
performed as described by Harris et al. (1982) using DNase I that had been calibrated with a rabbit
muscle G-actin standard. The presence of 1 % Triton has no effect on the inhibition of DNase by
actin (Blikstad et al. 1978).
RESULTS
Decoration of actin filaments with S-1
Actin filaments when decorated with the S-1 prepared for this study (Fig. 1) show
typical arrowhead morphology. Similarly, when microfilaments in demembranated
rat intestinal brush borders were treated with S-1 (Fig. 2), decoration resulted. The
ATPase activity of this S-1 was 0-9 /imol/min per mg protein, indicating a satisfactory
level of activity. This evidence, together with the typical arrowhead formations alluded to above and appropriate S-1 bands seen on SDS/acrylamide gels (Fig. 3),
confirm the reactivity of the myosin subfragment prepared and used in this work.
Disruption of cell and nuclear membranes
The best results were obtained by carrying out an initial prefixation with buffered
0-05% glutaraldehyde for 5 min followed by 0-01% Triton X-100 in buffer for
172
V. S. Hobbs, R. A. Jenkins andjf. R. Bamburg
Fig. 1. Rabbit skeletal muscle actin filaments decorated in vitro with myosin subfragment
S-l show typical 'arrowhead' configuration. X74000.
Fig. 2. S-l decorated actin filaments from microvilli of rat intestine. 'Arrowheads' point
towards the base of the microvilli and the terminal web. X 100000.
10 min. Cells so treated showed only minor distortions of gross morphology and cell
organelles, while cell and nuclear membranes were disrupted sufficiently (openings
of 3—80 nm) to provide adequate access for S-l into treated cells. Examples of the
effects of treatment are demonstrated in Fig. 4. When tannic acid was added to the
fixative it darkened the tissue, especially the microtubules and microfilaments.
Actin decoration not observed in S. teres
In spite of the obvious permeability of cells, and incubation of such treated cells
with S-l at a concentration high enough, and for periods sufficiently long, to allow
decoration to occur, no decorated microfilaments were observed in early dividing
micronuclei, metaphasemicronuclei(Figs4, 5) or later micronuclear stages. Filamentous material present within the dividing micronuclei (Fig. 5) did not appear to be
oriented with respect to microtubules or chromosomes and was often aggregated into
irregular clumps. At no time were any decorated filaments found in this material.
Furthermore, examination of the cortex of treated cells, with special attention to the
myonemal filament bundles believed to be involved in contractility, failed to reveal
decorated cytoplasmic filaments (Fig. 6).
Lack of actin in Spirostomum teres
M
173
A
Fig. 3. SDS/electrophoresis gels of myosin (M), actin (A) and S-l (S), prepared for this
study from rabbit skeletal muscle. The dark upper band on the myosin gel represents the
460 000Mr molecules, with the two heavy chains just beneath. The lower bands represent
the myosin light chains at 25 000, 18000 and 16000A/ r . On the actin gel the major band
is 46000A/ r . The S-l gel shows an upper 76 000MT band typical of the globular head and
two light chains at 21 000 and 18000Afr, lower on the gel.
DNase I inhibition assay
In a second effort to determine if either G or F-actin was present in these cells, we
utilized a DNase inhibition assay that is generally regarded to be specific for G-actin
(Blikstad et al. 1978; Harris et al. 1982). The large amount of protein released
following sonication of the cells indicated cell disruption, and the small pellet obtained
following high-speed centrifugation was completely soluble in the extraction buffer
containing 1 % Triton X-100. When 150[A of supernatant (4-5mg/ml, total protein)
from the S. teres extract was incubated with DNA no hydrolysis occurred, indicating
that no DNase activity was present in the extract. Triplicate analyses of the supernatant from the 5. teres extract that had been spiked with F-actin (16-7 fig/ml, final
concentration) before sonication gave values for G-actin of 16-8 ± 2-2^g/ml. These
results were calculated from the standard curve of G-actin inhibition of DNase I (Fig.
7). Thus, added F-actin seemed to be fully depolymerized by the extraction
procedure used and the resulting G-actin was stable in the presence of soluble protein
extract from S. teres at a final protein concentration of 4-5 mg/ml. Analysis of the
supernatant from the S. teres extract not spiked with actin failed to show any DNase
174
V. S. Hobbs, R. A. Jenkins andj. R. Bamburg
Figs 4 and 5
Lack of actin in Spirostomum teres
175
Fig. 6. Electron micrograph of a myoneme(m) from a tritonized S-1 treated cell. There is no
visible decoration of filaments within the bundle, however there are many mitochondria and
much smooth endoplasmicreticulum (ser) associated with the filament bundle. X32 500.
I inhibition when up to 180 fig of protein was used in the assay. When larger amounts
of extract were analysed some inhibition of DNase I was observed, but the inhibition
was significant only when 1 -45-2-9 mg of extracted protein was assayed. No DNase
I inhibitory activity was found in the Triton-solubilized pellet. The maximum actin
content of S. teres extract as a percentage of total protein was determined to be
0-06 ±0-01%.
Cell-model contractions
Triton-treated 5. teres cell models exposed to Ca 2+ /EGTA buffer solution in Ca2+
concentrations of 10~4 to 10~5 M underwent an immediate contraction of such magnitude that the morphology of the models was disrupted. In Ca2+ concentrations of
Fig. 4. Electron micrograph of a section of micronucleusfromS. teres treated with 0-01 %
Triton X-100, S-1 (2mg/ml) and 0-2% tannic acid added to the fixative. Disruption of
the nuclear envelope (arrows) appears sufficient to permit penetration of the S-1 subfragment. X52 500.
Fig. 5. High magnification electron micrograph of a metaphase micronucleus. Scattered
filamentous material (arrows) can be seen amongst the parallel microtubules. This cell was
treated with Triton X-100 and S-1; however, there is no evidence of arrowhead formation.
X 74 000.
176
V. S. Hobbs, R. A. Jenkins andjf. R. Bamburg
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80-
70-
£ 60
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2 50o
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40
30
20
10
0-5
10
1-5
20
G-actin (/zg/assay)
2-5
Fig. 7. G-actin inhibition of DNase I. A sample of G-actin (0-lmg/ml in extraction
buffer) was incubated with 2 jig of DNase I (SO/ig/ml) for 15 sand then 0-9 ml of a DNA
solution (100/^g/ml in 125mM-Tris-HCl, 5mM-MgCl2, 2mM-CaCl 2 , pH7-8) was
added. The linear initial rate of absorbance increase at 260 nm was measured and compared to the rate with no actin added.
10~5 to 5 X 10~6M the results were similar but contraction was slower, while lower
concentrations caused no change in morphology. Also, Ba2+, Mg2"1" and Sr2"1" at concentrations up to 10~ 4 M, caused absolutely no contraction. No ATP was required for
the above reactions and reversal was not possible because of the extent of contraction.
DISCUSSION
The lack of evidence for actin in 5. teres is surprising, in view of the wide variety
of cells in which actin is known to be an integral cytoplasmic and/or nuclear protein.
Lack ofactin in Spirostomum teres
177
In general, the procedures used in this work were designed to stabilize and preserve
any actin filaments present. The pH range for initial isolation and treatment of 5. teres
(pH 6-8-7-2) should preserve microtubules and microfilaments. Triton stabilizes
actin microfilaments (Nagata, Sagara & Ichikawa, 1980), and acetone dehydration,
unlike ethanol, does not cause shortening of actin thin filaments in muscle fibres (Page
& Huxley, 1963) and also seems preferable for dehydration of non-muscle tissue.
Maupin-Szamier & Pollard (1978) reported destruction of actin filaments by 40 mM
-osmium in 50 mM-cacodylate buffer (pH7'3) at 25 °C; however, the use of tannic
acid (0-2 %) is reported to prevent such disruption and to preserve the microfilaments
better (Begged al. 1978; Seagull & Heath, 1979). In this study no actin filaments were
observed in S-l/tannic acid-treated cells (Fig. 4).
There is no doubt that filamentous material can be seen scattered among the
microtubules in the micronuclei of dividing S. teres. But this appears to be distributed
randomly throughout the nucleoplasm and does not label with S-l. Likewise, clearly
observed cytoplasmic thin filaments remain undecorated in 5. teres, whereas similar
filaments in many other cells have been identified as actin (Forer 1978a,b; Korn,
1978; Pollard & Weihing, 1974; Sanger & Sanger, 1980).
The DNase I inhibition assay is believed to be specific for G-actin (Blikstad et al.
1978), although other proteins that bind to DNase have been reported (Kohama &
Holtzer, 1981). Assays performed at very high protein concentration may show inhibition due to substrate availability, if DNA binding proteins are present. This assay
was used in the present work to help determine the presence or absence of actin in S.
teres. Forms of actin produced by membrane interaction with muscle G-actin, and
which fail to inhibit DNase I, have been reported (Grazief al. 1980). However, this
modified actin also fails to assemble under standard conditions of actin assembly,
which implies that it is denatured; apparently this modified actin will assemble under
the influence of added filamentous myosin. Since S. teres may contain lytic enzymes
within digestive vacuoles, there was the possibility that cell lysis might release
enzymes that could degrade or modify the actin so that it would no longer inhibit
DNase. Alternatively, endogenous DNase may be released, which could bind to the
actin present and alter the results of the assay. However, no endogenous DNase
activity was found in the S. teres extracts and actin added to the extracts was stable
and could be measured using this assay.
Another possible reason for not detecting actin in 5. teres extracts is that a form of
actin that does not inhibit pancreatic DNase I exists in this organism. Such an 'actinlike' protein has been described in extracts of Entamoeba histolytica (Gadasi, 1982).
However, actins from other eukaryotic microorganisms such as Acanthamoeba
(Reichstein & Korn, 1979) and Dictyostelium (J. D. Pardee, personal communication) inhibit DNase I to the same degree as actins from mammalian sources. In
addition, even the unusual actin from E. histolytica bound to and activated rabbit
muscle myosin (Gadasi, 1982), so that one would expect the S-l decoration studies
to have confirmed the presence of actin filaments.
Actin forms, other than individual 6-8 nm microfilaments, have been observed,
including nets (Hinssen, 1972; Spudich & Cooke, 1975) and bundles (Stossel &
178
V. S. Hobbs, R. A. Jenkins andj. R. Bamburg
Hartwig, 1975); such supramolecular aggregates were not found in S. teres. Forer
(197&3,6) also points out that actin can exist in cells in forms that will not interact with
HMM (and presumably S-l), and such a possibility cannot be overlooked in the
present study. However, in view of the results from the DNase I inhibition assay, it
seems more probable that this c"ell makes use of a contractile protein other than actin.
Routledge (1978) glycerinated the spasmonemes of vorticellids and showed that
they contract in the presence of calcium ions ( 1 0 ~ 5 M ) and extend when the calcium
concentration is lowered to 10~ 8 M. A protein of 20000 molecular weight extracted
from the spasmoneme was found to have a high affinity for calcium binding. Routledge suggested a functional relationship between such proteins (spasmins) and the
calcium-binding proteins of striated muscle, which behave similarly. However, the
spasmoneme contraction does not require ATP and is not inhibited by ATPase inhibitors.
A similar mechanism may operate in S. teres. Ettienne (1976) reported regulation
of contractility in Spirostomum by the action of Ca ; extracted cell models were
found to contract when the calcium concentrations were greater than 10~5 M. Similar
results were obtained in this study, although relaxation was not possible because of
the extent of contraction.
In conclusion, this study has provided evidence that suggests that actin is not
involved in the motile processes in 5. teres. It may be argued that although actin was
not found in the mitotic apparatus of this organism by S-l decoration, it may still be
present in a non-filamentous form, or that the cells were not sufficiently permeable
for S-l penetration. The results of the DNase I inhibition assay certainly moderate
such arguments and place the maximum actin concentration at 0-06 % of the total
soluble protein. An actin-based contractile system also seems to be ruled out. It does
seem probable, however, that this contractile ciliate has a calcium-sensitive, spasminlike contractile protein system similar to that described for the vorticellid spasmoneme.
This work was supported in part by grant number NIH GM23878 from the National Institute
of Health to Robert A. Jenkins. DNase I inhibition assay supported by grant number NS 10429 from
the National Institute of Neurological and Communicative Disorders, and Stroke, to J. R. Bamburg.
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{Received 4 January 1982-Revised 31 August 1982)