Download cilia regeneration in tetrahymena and its inhibition

CILIA REGENERATION IN TETRAHYMENA
AND ITS INHIBITION BY COLCHICINE
JOEL L. ROSENBAUM and KATHRYN CARLSON
From the Department of Biology, Yale University, New Haven, Connecticut 06520
ABSTRACT
The cilia of Tetrahymena were amputated by the use of a procedure in which the cells remained viable and regenerated cilia. Deciliated cells were nonmotile, and cilia regeneration
was assessed by scoring the percentage of motile cells at intervals following deciliation. After
a 30-min lag, the deciliated cells rapidly recovered motility until more than 90% of the cells
were motile at 70 min after amputation. Cycloheximide inhibited both protein synthesis
and cilia regeneration. This indicated that cilia formation in Tetrahymena was dependent on protein synthesis after amputation. Conversely, colchicine was found to inhibit
cilia regeneration without affecting either RNA or protein synthesis. This observation suggested the action of colchicine to be an interference with the assembly of ciliary subunit
proteins. The finding that colchicine binds to microtubule protein subunits isolated from
cilia and flagella (13) supports this possibility. The potential of the colchicine-blocked ciliaregenerating system in Tetrahymena for studying the assembly of microtubule protein subunits during cilia formation and for isolating ciliary precursor proteins is discussed.
INTRODUCTION
The elongation of ciliary buds in cultured fibroblasts is inhibited by the colchicine derivative
colcemid (16). It is possible that this drug inhibits
ciliogenesis by binding to the ciliary microtubule
precursors, thus preventing their assembly. This
suggestion is reinforced by the finding that colchicine binds in vitro to subunit proteins isolated
from ciliary and flagellar microtubules (13), and
that it causes the disruption of microtubules of
other organelles in vivo by a mechanism which
also seems dependent on an affinity for microtubule subunits (1, 2, 7, 17-19).
Systems of regenerating cilia and flagella in the
protozoans have been shown to offer many advantages for the investigation of various aspects of the
synthesis and assembly of ciliary and flagellar
microtubules (3, 11, 12). It was of interest to determine the effect of colchicine on the regeneration
of cilia in the protozoan Tetrahymena. If inhibition
of cilia regeneration in the presence of colchicine
occurred, and if this inhibition could be shown to
result from the binding of colchicine to ciliary
microtubule precursor proteins, the use of tritiated
colchicine (1) in this system would permit the isolation of ciliary microtubule subunits from regenerating cells. One could then proceed with the
biochemical characterization, investigation of
turnover rates, and the study of the in vitro
assembly of these precursor proteins.
The present report will show that both colchicine and the protein synthesis inhibitor, cycloheximide, inhibit the regeneration of Tetrahymena
cilia, but that only colchicine has this effect without altering either protein or RNA' synthesis.
These results are consistent with the hypothesis
that colchicine is interfering with cilia elongation
in Tetrahymena by preventing the assembly of
ciliary microtubule subunits.
'1Abbreviations: RNA, ribonucleic acid; EDTA 2
Na, disodium salt of ethylenediamine tetraacetic
acid; TCA, trichloroacetic acid.
415
FIGUs
1 Cilia amputation in Tetrahymena pyriformis (W). Fig. 1 a. A typical ciliated cell before amputation or after complete cilia regeneration; Fig. 1 b. A completely deciliated cell characteristic of most cells
after amputation and before regeneration. Fig. 1 c. Cell after amputation, with oral cilia still attached but
immotile. These cilia will drop off before regeneration. Cells were fixed for 3 days in 2.5% glutaraldehyde
°
in 0.05 M cacodylate buffer (pH 7.0) at 4 C. Photomicrography with Zeiss 40 X Apo oil (phase) objective
with mercury lamp and dark-field illumination. (Zeiss Ultra condenser, Carl Zeiss, Inc., New York, N.Y.).
X 700.
MATERIALS AND METHODS
Assay for Cilia Regeneration
Culture Conditions
Since deciliated cells are nonmotile, regeneration
could be assessed by determining the percent of
motile cells at various times after the amputation
procedure. A 0.8 X 32 mm micro-hematocrit tube
(Scientific Products Co., Evanston, Ill.) was filled by
capillarity with the incubated cell suspension. The
percent of moving cells was scored by placing the tube
under heavy mineral oil, and observing the cells in
the tube with a binocular dissecting microscope (40 X
magnification). Only those cells with definite movements were scored as motile. At least 150 cells were
observed in each sample.
Tetrahymena pyriformis (W) was grown in defined
medium (6). 125 ml of medium in a 1000 ml Roux
flask were inoculated with 2.5 ml of a 2-day-old culture, and the organisms were allowed to grow for 2
0
days at 25 C. The cell density at this time was 5-7 X
105 cells/mi.
Cilia Amputation Procedure
The cells were harvested at room temperature
(23°C) by centrifugation at 160 g for 3 min, and were
concentrated and resuspended in their original growth
medium (final concentration, 2 X 106 cells/mi). The
concentrated cells were used immediately for the
deciliation procedure which was carried out at 2 °C. At
zero time, 2.5 ml of the concentrated cells were added
to 5.0 ml of medium A (10 mM EDTA 2 Na; 50 mM
sodium acetate, pH 6.0) in a 50 ml conical centrifuge
tube and mixed by swirling. At 30 sec, 2.5 ml of cold
distilled water was added, followed by the addition at
90 sec of 0.25 mil of 0.2 M CaCl2. After the addition of
calcium chloride, the suspension was mixed by inverting the tube several times. At 3 min and 30 sec, the
suspension of cells was subjected to 2-4 shearings with
a 10 ml glass syringe fitted with an 18 gauge needle.
Immediately after deciliation (about 4 min), one
volume of the suspension was pipetted into 20 volumes
of recovery solution (fresh defined growth medium or
0
0.01 Mpotassium phosphate buffer, pH 7.0) at 25 C.
The deciliated cells were then incubated at 25 0 C
with gentle agitation, and cilia regeneration was assayed as described below.
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Determination of In Vivo RNA and
Protein Synthesis
The in vivo incorporation of 14C-amino acids and
'4 C-uridine into total Tetrahymena protein and RNA,
respectively, was assayed by the filter disc method (8).
Biochemicals
Cycloheximide (Acti-dione) was obtained from
The Upjohn Co., Kalamazoo, Mich., and from
Nutritional Biochemical Corporation, Cleveland,
Ohio. Colchicine was also obtained from Nutritional
Biochemicals. Most of the work in this report was
carried out with one lot number of colchicine. However, different lot numbers were shown to have somewhat varying potencies for the inhibition of cilia
regeneration. It was necessary, therefore, to run a new
dose-response curve for the inhibition of cilia regeneration for each new lot of colchicine. Colcemid was
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2 The kinetics of recovery of motility (cilia regeneration) in deciliated Tetrahymena. Squares, open
circles, closed circles, and triangles represent four separate experiments and show the reproducibility of the
assay for recovery of motility after cilia amputation.
FIGURE
purchased from CIBA Pharmaceuticals Co., Summit, New Jersey.
RESULTS
The Cilia-regeneratingSystem
THE DECILIATION
PROCEDURE:
The con-
tinuity of the ciliary membrane with the cell membrane in Tetrahymena creates difficulties in design-
ing a procedure for the removal of cilia which
maintains both the viability of the cells and their
ability to regenerate cilia. Detachment of all cilia
usually causes lysis or, in nonlysing cells, failure to
regenerate new cilia. These problems are circumvented by modifying the cilia isolation procedure
of Watson et al. (20), omitting ethanol from the
deciliation solution, lowering the pH, and adding
a mechanical shearing step to remove immobilized
cilia. This modified procedure results in deciliated
cells which, although fragile, maintain their normal pyriform shape and regenerate cilia (Fig.
l a-c).
Phase microscope observations of deciliated cells
indicate that the majority of the cells have lost all
of their somatic and oral cilia (Fig. Ib). A few
cells usually retain some oral cilia after the amputation procedure (Fig. Ic), but these cilia drop off
after the cells have been in the recovery solution
for about 10 min. Electron microscope observations
of cells after cilia amputation and during regenera-
JOEL L. ROSENBAUM AND KATHRYN CARLSON
Inhibition of Cilia Regeneration by Colchicine
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Effect of colchicine and colcemid concentration on cilia regeneration in Tetrahymena. Recovery
of motility was assayed at 90-min postamputation. Circles, colcemid; triangles, colchicine.
FIGURE 3
tion2 indicate that the cilia detach at the distal
portion of the basal body.
CILIA REGENERATION:
In Fig. 2 it can be
seen that the first cells have recovered their motility by 30-35 min after amputation; this recovery
is coincident with the appearance of ciliary shafts.
The population then rapidly regains motility until
60-70 min after deciliation when more than 90%
of the cells are motile. Observations of single cells
during the recovery period indicate that the oral
cilia grow out first and elongate synchronously,
while the somatic cilia grow out later and elongate
asynchronously over the cell surface.
Since the cells are motile when only part of their
ciliary complement is regenerated, an assay based
on the percentage of cells moving at any time is
not completely representative of either the number
or the length of the cilia on each organism. Phase
and electron microscopic observations show this
discrepancy. While the full complement of mature
2
Joel L. Rosenbaum. Unpublished results.
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THE JOURNAL OF CELL BIOLOGY
cilia is not present until almost 100 min after
amputation, the assay shows the population to be
fully motile by 60-70 min postamputation. However, the kinetics of recovery are reproducible and
afford a quick and convenient method for assessing
the over-all cilia formation in a population.
The Effect of Colchicine on Cilia Regeneration
INHIBITION
OF
ELONGATION:
The
re-
covery of deciliated cells is completely inhibited
when 4 mg/ml colchicine or 0.5 mg/ml colcemid
is included in the recovery medium (Fig. 3). This
inhibition of recovery is not due to an inhibition of
motility since phase microscope observations indicate the absence of ciliary shaft regeneration in the
presence of colchicine or colcemid.
The concentration of colchicine required for the
inhibition of cilia regeneration is high but is
found not to immobilize normal ciliated cells.
Furthermore, this concentration has no observable effect on either RNA or protein synthesis in
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FIGURE 4 Effect of colchicine on uridine-' C incorporation into Tetrahymena RNA. Uridine-2-14C
(specific activity, 43 mc/mmole; concentration, 5 uc/ml cells) was added at zero time and the colchicine
(4 mg/ml) at 0 min (arrow). Circles, control; triangles, colchicine.
Tetrahymena during the time course of the experiment (Figs. 4 and 5), or on the oxygen consumption of control and colchicine-treated cells. There
is no effect on the recovery of motility when a
concentration of sucrose, equimolar to the colchicine concentration, is added to the recovery
solution. Deciliated cells maintain their normal
pyriform shape and the contractile vacuole functions in the presence of colchicine.
Colchicine acts rapidly in inhibiting cilia regeneration. Addition of the drug to the recovery
solution when cells are just beginning to recover
their motility (40 min postamputation) completely inhibits recovery within 10 min (Fig. 6).
RECOVERY
FROM
COLCHICINE
INHIBI-
Fig. 7 shows the recovery kinetics of
deciliated cells which have been diluted into fresh
recovery medium after varying lengths of time in
4 mg/ml colchicine. After the removal of colchicine, motility is regained by normal recovery
TION:
JOEL L. ROSENBAUM AND KATHRYN
kinetics following the usual 30-40 min lag; this
recovery is independent of the length of time (at
least until 250 min) in colchicine.
In the presence of 4 mg/ml colchicine, deciliated cells will remain without cilia for about 3-5
hr and then begin recovery in the continued
presence of colchicine. The cells will eventually
regain full motility, although the recovery kinetics
are slower than normal. Higher concentrations of
colchicine (8-12 mg/ml) will extend the duration
of the inhibitory period. Many vacuoles with yellow crystals can be observed in the cells coincident
with the recovery. If cells recovering from the
colchicine treatment are placed in fresh colchicine,
they will continue to recover. This recovery occurs
even if the recovering cells are redeciliated and
placed in fresh colchicine. Thus, the cells may
not only be able to concentrate the colchicine in
vacuoles, but they may also become resistant to
colchicine after exposure to it. Studies on the up-
CARLSON
Inhibition of Cilia Regeneration by Colchioine
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Effect of colchicine on amino acid-' 4 C incorporation into TCA-precipitable protein of Tetrahymena. Amino acid mixture- 14C (concentration, S c/ml cells) was added at zero time and the colchicine
(4 mg/ml) at 15 min (arrow). Circles, control; triangles, colchicine.
FIGURE 5
take of colchicine- 3H by Tetrahymena2 have helped
to illuminate this problem. The results indicate
that at about the same time the cells are overcoming colchicine inhibition, the concentration of
colchicine-'H which has been taken up begins to
decrease. It is relevant to point out here that no
such spontaneous recovery from colchicine inhibition of elongation occurs when similar experiments are carried out with the flagella-regenerating system in Chlamydomonas; the cells remain without flagella as long as colchicine is present. 3
Therefore, the overcoming of colchicine inhibition
in Tetrahymena is probably a unique phenomenon
related to the feeding and excretory mechanisms
of this organism and will require further studies
with colchicine- 3 H.
Rosenbaum, J. L., J. Moulder, and D. Ringo. In
preparation.
3
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The Effect of Cycloheximide on
Cilia Regeneration
Since colchicine inhibited cilia regeneration
without affecting protein synthesis, it was of interest to determine the effect of an inhibitor of
protein synthesis on cilia regeneration. In Fig.
8 it can be seen that 10 ug/ml cycloheximide, a
concentration sufficient to inhibit amino acid
incorporation into TCA precipitable protein of
Tetrahymena (Fig. 9), prevented deciliated cells
from recovering motility in the phosphate buffer
recovery solution.
DISCUSSION
The cilia-regenerating system described in this
report offers several advantages for investigating
problems concerned with the synthesis and assembly of ciliary proteins: (a) Tetrahymena can be
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FIGURE 6 Effect of time of addition
of colchicine on cilia regeneration in
Tetrahymena. Colchicine (4 mg/ml)
was added at zero time (open squares),
10 min (open circles), 20 min (closed
triangles), 30 min (open triangles)
and 40 min (closed squares); closed
circles, control.
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grown in large quantities in a completely defined
medium (6), (b) its division can be easily synchronized (9), and (c) methods are available for
isolating the cilia (20) and fractionating them into
component parts (4).
Results obtained with the use of this system
indicate cilia regeneration to be dependent on
protein synthesized after amputation. Thus, cycloheximide inhibits both protein synthesis and cilia
regeneration. This result does not permit us to
make an unequivocal statement about the amount
of ciliary precursor pool in the cells, since the assay
for cilia regeneration only measures the recovery
of motility of deciliated cells. Therefore, a small
amount of ciliary growth, insufficient to impart
motility to the cycloheximide-inhibited cells,
would not be apparent. Although phase and dark
field microscope observation of cycloheximideinhibited cells shows the absence of ciliary stubs, a
more accurate assessment of the treated cells
could be made with the use of the Protargol
staining procedure for ciliated protozoans (21)
and with electron microscopic observations.
Studies on flagellar regeneration in Chlamydomonas have suggested the existence of a flagellar
JOEL L. ROSENBAUM AND KATHRYN
precursor pool which varies in amount in different
mutant strains.3
Colchicine inhibits cilia regeneration apparently without affecting RNA or protein synthesis,
and it is suggested that colchicine acts in this
system by interfering with the assembly of ciliary
microtubule subunits. This possibility is strengthened by studies on the mode of action of colchicine
carried out in Taylor's laboratory (1, 2, 13, 17).
He found that a concentration of colchicine sufficient to inhibit mitosis had little effect on RNA
and protein synthesis and, by analyzing the
kinetics of colchicine- 3 H uptake and binding, he
was able to hypothesize that colchicine was blocking mitosis by binding to the subunits composing
the mitotic tubules (17). This hypothesis was subsequently verified by the demonstration that colchicine will bind quite specifically to microtubule
subunits isolated from various sources, including
the mitotic spindle and flagellar axonemes (1, 2,
13).
The results presented on the reversibility of
colchicine inhibition show that even after prolonged blocking of regeneration with colchicine,
the rate of recovery of motility in fresh medium is
CARLSON
Inhibition of Cilia Regeneration by Colchicine
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FIGURE 7 Recovery of motility in deciliated cells after removal of colchicine. Colchicine (4 mg/ml) was
added at zero time and the cells were removed from colehicine at 20 min (open triangles), 30 min (open
circles) and 50 min (closed triangles); closed circles, control.
the same. If the cells are synthesizing ciliary protein during the block, and if the amount of precursor is the rate-limiting step in recovery of
motility, the rate should be faster and/or the lag
period shorter. The fact that the rate is not faster
after release from colchicine inhibition suggests
that the assembly step in ciliary elongation may
be rate-limiting. That this is probably so has
been shown in experiments (to be reported) with
the (,lamydomonas flagella-regenerating system.3
These results indicate that the amount of flagellar
elongation in cycloheximide but not the rate can
be increased by treatment of deflagellated cells
with colchicine prior to washing and placing the
cells in cycloheximide.
A precautionary note should be added to the
interpretation of the inhibitor results presented
in this report. First, although the data show that
cycloheximide will almost totally inhibit amino
acid incorporation into the TCA-precipitable
protein of Tetrahymena, they do not exclude the
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THE JOURNAL OF CELL BIOLOGY
possibility that cycloheximide is inhibiting the
synthesis of some nonciliary protein required for
regeneration. However, a forthcoming paper re-porting results obtained with the Chlamydomonas
flagella-regenerating system 3 treats this topic
more thoroughly and indicates that this inhibition
of nonciliary protein synthesis probably is not the
case. Second, although the amino acid incorporation data indicate that colchicine is not blocking
protein synthesis, an inhibition of about 1% would
not be apparent with the accuracy of the procedure. This amount could be critical since the
cilia comprise about 1% of the total protein, and
it is conceivable that colchicine specifically
inhibits ciliary protein synthesis. Some of the experiments described below will be necessary to
define this latter problem more thoroughly.
The colchicine-3 H binding assay for microtubule subunit proteins described by Borisy and
Taylor (1) provides a method for directly determining whether or not the colchicine inhibi-
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FIGURE 8
Effect of cycloheximide on
the incorporation of amino acid-14 C
into TCA-precipitable protein of
Tetrahymena. Amino acid mixture-' 4C
(concentration, 3 c/ml) was added
at zero time and cycloheximide at 15
min (arrow). Closed circles, no cycloheximide; triangles, 1 ig/ml; open
circles, 10 jig/ml.
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JOEL L. ROSENBAUM AND KATHRYN
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Inhibition of Cilia Regeneration by Colchicine
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FIGURE 9 Effect of cycloheximide concentration on
cilia regeneration in Tetrahymena. Cycloheximide in
varying concentrations was added to the cells at the
time of amputation; the recovery of motility in phosphate buffer recovery medium was assessed at 90 min
postamputation.
tion of cilia regeneration described in this report
is caused by the binding of colchicine to the ciliary
microtubule subunits. We are currently using this
assay (a) to analyze the kinetics of colchicine-3H
uptake and binding in Tetrahymena, (b) to determine if there is an increase in colchicine-binding
protein in regenerating cells over that in nonregenerating cells, as is suggested by the cycloheximide inhibition studies, and (c) to isolate
ciliary microtubule precursor proteins from colchicine-3H blocked cilia-regenerating systems.
There have been several studies on the chemistry of microtubules in recent years (2, 5, 10, 1315), but most of these investigations have been
carried out on subunits isolated from assembled
tubules (see, however, reference 2). It would be
important to isolate the precursor proteins before
their time of assembly and to compare their biochemical characteristics with those of subunits
isolated from assembled tubules. The isolation of
these precursor proteins would permit one to
attack such problems as the turnover rate of the
ciliary microtubule precursors, the time of attachment of the guanine nucleotide to the subunits,
and the possible role of the nucleotides in the polymerization of subunits (14).
Finally, observations on ultrastructural changes
in Tetrahymena during (a) normal regeneration,
(b) the period during colchicine inhibition when
cells remain without cilia for a few hours, and (c)
the period when cells are overcoming the colchicine inhibition, are feasible with this cilia-regenerating system. These problems are currently
under investigation and offer a new approach for
the study of ciliogenesis.
We would like to thank Dr. F. M. Child (Trinity
College, Hartford, Conn.) for his assistance in designing the cilia-regenerating system in Tetrahymena,
Professor H. Swift (University of Chicago, Chicago,
Ill.) for making his electron microscope facilities available for preliminary observations on the ultrastructural changes occurring during cilia regeneration,
and Miss Joanna Olmsted for editorial aid.
This investigation was supported by grants No. GM14642 from the United States Public Health Service
and IN-31-H-908 from the American Cancer Society
to Joel L. Rosenbaum.
Received for publication August 1968, and in revised form
24 September 1968.
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