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Hormonal regulation of actin and tubulin in an epithelial cell line from Chironomus tentans.

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Archives of Insect Biochemistry and Physiology 46:11–18 (2001)
This article originally published in Volume 41
Archives of Insect Biochemistry and Physiology 41:71–78 (1999)
Hormonal Regulation of Actin and Tubulin in an Epithelial
Cell Line From Chironomus tentans
A. Fretz and K.-D. Spindler*
Abteilung Allgemeine Zoologie, Universität Ulm, Ulm, Germany
The morphogenetic changes in an epithelial cell line from
Chironomus tentans that are evoked by molting hormones and
molting hormone agonists are accompanied by transient changes
in the concentration of actin and b-tubulin protein and mRNA.
As compared to controls, actin protein and mRNA concentrations
increase by about 50%, whereas tubulin reaches maxima of 100%
increase. The proportion between globular and filamentous actin remains constant after hormone treatment. Arch. Insect
Biochem. Physiol. 41:71–78, 1999. © 1999 Wiley-Liss, Inc.
Key words: cytoskeletal proteins; 20-hydroxyecdysone; Chironomus tentans;
cell-line
INTRODUCTION
The epithelial cell line from Chironomus
tentans, established in 1982 by Wyss, grows exclusively as multicellular monolayered vesicles.
This cell line responds to molting hormones as
already noticed by Wyss (1982) and summarised
in a recent review (Spindler-Barth and Spindler,
in press). An ecdysteroid receptor (EcR) has been
demonstrated in this cell line both by investigations on hormone binding (Turberg et al., 1988;
Turberg and Spindler, 1992), by the isolation of
an EcR gene (Imhof et al., 1993), and the demonstration of both heterodimerization partners—
EcR and USP—in the cells by immunological techniques (Lammerding-Köppel et al., 1998; Rauch
et al., 1998). Due to nearly non-existing hormone
synthesis and metabolism (Spindler and SpindlerBarth, 1991), this cell line is well suited for studies on molting hormone action (Dinan et al., 1990).
When the cells are incubated with molting
hormones, proliferation stops, followed by an initiation of cell differentiation (summarised in
Spindler-Barth and Spindler, in press). Early
events in this hormonally induced morphogenetic
process are a reduction in DNA (Spindler et al.,
© 2001 Wiley-Liss, Inc.
1993) and protein-synthesis (Fretz et al., 1993).
Both mRNAs (Fretz et al., 1993) and proteins
(Fretz et al., 1993; Quack et al., 1995) change in
a complex and time-dependent manner after addition of molting hormones. In addition to these
general effects of molting hormones, changes in
the activities of some enzymes related to cuticle
formation and morphogenesis, as well as changes
in the concentrations of the muscarinic acetylcholine and ecdysteroid receptors, as well as in the
pattern of phosphorylation of USP were demonstrated (reviewed by Spindler-Barth and Spindler,
in press). Since molting hormones induce a drastic change in cell shape and a reorientation of
microtubules in this cell line (Spindler-Barth et
Abbreviations used: EcR = ecdysone receptor; PCR = polymerase chain reaction; USP = ultraspiracle receptor.
A. Fretz’s present address is Institut für Medizinische
Biochemie, Universität Rostock, D-18057 Rostock, Germany.
*Correspondence to: K.-D. Spindler, Abteilung Allgemeine
Zoologie, Universität Ulm, D-89069 Ulm, Germany. E-mail
address: Klaus-Dieter.Spindler@Biologie.Uni-Ulm.de
Received 30 July 1998; accepted 15 November 1998
12
Fretz and Spindler
al., 1992), an investigation of cytoskeletal elements seemed to be worthwhile. Furthermore, the
morphogenetic response of Kc-cells from Drosophila melanogaster is also evoked by molting hormones (Courgeon 1972), and these morphological
changes are accompanied by changes in actin
(Couderc et al., 1982) and tubulin (Montpied et
al., 1988) concentrations. Because of the different morphogenetic responses in C. tentans and
D. melanogaster cells, a comparison of the underlying mechanisms was necessary.
MATERIALS AND METHODS
Cell Culture
The epithelial cell line from Chironomus
tentans was cultured according to Wyss (1982).
Cells were subcultured every 10–12 days (split
ratio 1:10–1:20) after dispersing the multicellular vesicles by pipetting. For hormone treatment
1 µM 20-OH-ecdysone (final concentration) was
added to the culture medium 7 days after dispersion, except for experiments in Figure 3, where
9-day-old cells were used.
Densitometric Determination of Proteins
and RNA
All final signals (silverstained protein, chemiluminescence signal after Western blot, ethidium
bromide stained nucleic acids, X-ray films, methylene blue stained RNA) were scanned (Scanner JX325, Sharp, 600 dpi, software ViceVersa Scan 1.2,
Krystec EDV, Norderstedt, Germany) and analyzed
with an image analysis system (PHORETIX, Nonlinear Dynamics Ltd, Newcastle, UK; resolution:
600 dpi, corresponding 42 µm2). Calibration curves
were determined for each signal and sample. The
intensity of a given band was then quantified and
taken as a measure for concentration.
Western Blots
Total protein lysates were separated on SDSpage according to Laemmli (1970). Fifty micrograms
protein/lane was loaded onto 10 % SDS-polyacrylamide gels (0.6x MDE Gel Solution [Böhringer,
Ingelheim, Germany], Minigel Twin, 8.6 × 7.2 × 0.1
cm [Biometra, Göttingen, Germany]). Protein was
determined according to Bradford (1976) using bovine serum albumin as standard. Western blots
were performed according to Khyse-Andersen
(1984). After semi-dry electroblotting, the nitrocellulose membranes (BA 85, 45 µm pore size, Schleicher & Schuell, Keene, NH) were soaked in blocking
buffer (5% milk powder, 1% fat, in 10 mM Tris/HCL,
pH 7.5, containing 150 mM NaCl and 0.05% Tween
20). The protein blot was probed with a monoclonal
antibody against sea urchin β-tubulin (CalbiochemNovabiochem) diluted 1:100 in blocking buffer. The
secondary antibody (anti-Mouse IgG, peroxidase
conjugated; Sigma, St. Louis, MO) was diluted
1:1,000 (10 mM Tris/HCL, pH 7.5, containing 150
mM NaCl and 0.05 % Tween 20). Protein bands
were visualised using an ECL detection kit (Amersham, Arlington Heights, IL) according to the instructions of the supplier. The reaction product of
the peroxidase coupled to the second antibody shows
chemiluminescence, which was recorded on X-ray
film (Biomax, Kodak, Rochester, NY).
Determination of Globular and
Filamentous Actin
Since two antibodies against actin from
Amoeba proteus (monoclonal, Sigma) and vertebrates (monoclonal, Amersham) gave no positive
signals in Western blots, even if 50 µg protein
per lane was separated, actin had to be quantified by the DNase inhibition assay according to
Blikstad et al. (1978) with slight modifications.
The assays were performed in triplicates. Cells
from 1.5 ml cell culture were pelleted, washed
and lysed in a 5 mM Tris/HCl buffer, pH 7.5,
containing 150 mM NaCl, 2 mM MgCl2, 0.1 mM
DTT, 0.2 mM ATP, and 0.5% Triton X 100. Of
this solution 5 and 10 µl were used for the assays, containing 10 µl of DNase solution (0.1 mg/
ml DNase I, DN 100, Sigma, 50 mM Tris/HCl,
pH 7.5, 0.5 mM CaCl2, and 0.01 mM PMSF) and
480 µl DNA solution (40 µg calf thymus DNA
typ I, Sigma per ml, 100 mM Tris/HCl, pH 7.5,
4 mM MgSO4, 1.8 mM CaCl2). The decrease of
DNA concentration without addition of actin
leads to a decrease in absorption of 0.08 to 0.1
per min at 260 nm, which is inhibited by the
addition of actin (20 to 140 ng). Since only globular actin interacts with DNase, determination
of filamentous actin can be performed only indirectly. Total actin in the samples was depolymerized (addition of the same volume of a buffer
containing 20 mM Tris/HCl, pH 7.5, 1.5 M
guanidinium-HCl, 1 mM sodium acetate, 1 mM
Actin and Tubulin in Chironomus tentans
CaCl2, 1 mM ATP), the sample was measured
again, and the difference between the two measurements was due to filamentous actin. Since
in contrast to the original paper on this method
(Blikstad et al., 1978) and a similar investigation in Drosophila (Couderc et al., 1982), actin
concentration was not stable over prolonged time
periods despite the addition of higher concentrations of PMSF and of other protease inhibitors,
careful kinetic analyses were necessary to determine accurately the actin concentrations in
our cell lysates (Fig. 1).
13
Polymerase Chain Reactions
Cells (10 days old) were collected by centrifugation (1,000g, 3 min) and washed once with PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Na-phosphate
buffer, pH = 6.8). RNA was isolated according to
Chomczynski and Sacchi (1987) using TRIzolTM reagent (Gibco, Gaithersberg, MD). Messenger RNA
was obtained with OligotexTM (Qiagen, Hilden, Germany) according to the manufacturer.
PCR were performed in an Omnigene temperature cycling system (Hybaid, MWG). cDNA templates were synthesised with 5 µg total RNA, 5 ng
oligo(dT) primer and 200 U M-MLV transcriptase
(Gibco, Gaithersburg, MD) using the degenerate oligonucleotides ACT U235: 5´- AA(C/T)TGGGA(C/
T)GA(C/T)ATGGA(A/G)AA - 3´ (sense) and ACT
L667: 5´- GCCAT(C/T)TC(C/T)TG(C/T)TC(A/G)AA(A/G)TC - 3´ (antisense) located in the conserved region of actins from protozoa, insects,
tunicates and mammals and the degenerate oligonucleotides TUB U 790 5′ - CA(C/T)TT(C/
T)TT(C/T)ATGCC(N)GG(N)TT- 3′ (sense) and
TUB L 1203 5′ – A(C/T)TCCAT(C/T)TC(A/G)TCCAT(N)CC – 3′ located in the conserved region
of β-tubulins from bacteria, plants, protozoa, insects, Xenopus laevis, chicken, and human. A 50µl
reaction mixture contained (final concentrations)
7 mM Tris-HCl, pH 8.4, 35 mM KCl, 0.02 mM
dNTP-Mix, 0.08 mM MgCl2, 0.2 µg of each primer,
Fig. 1. Quantitative determination of actin. A: Between 20
and 140 ng, there is a linear relationship between actin concentration and inhibition of DNase. B: G-actin concentration (open symbols) in the extracts was determined at
different times after lysis. After 20 min, a part of each sample
was treated with guanidine hydrochloride and the total
amount of actin (filled symbols) was determined. The difference between the two lines (----) represents F-actin.
Isolation of RNA
14
Fretz and Spindler
cDNA as template and 1 unit Taq-DNA-polymerase (Gibco, Gaithersburg, MD). The enzyme
was added after an initial denaturation step (5
min, 93°C) annealing (90 sec, 49–58°C), and extension (2 min, 72°C), followed by a final extension step (7 min, 72°C). The PCR product was
separated on a 1.5% agarose gel. The amplification products were directly sequenced with the
Sequenase Version 2.0 PCR Product Sequencing
Kit (USB, Cleveland, OH) according to the manufacturer.
hering sperm for 3 h at 55°C and then hybridized with the 32P-labelled (Megaprime DNA labelling system, RPN 1607, Amersham) probes in
6 × SSC, 0.5% SDS, and 0.01% DNA for 18 h at
55°C. Filters were washed twice with 2 × SSC,
0.1% SDS for 20 min, and then 3 times with 0.5
× SSC, 0.1% SDS for 30 min at 68°C. Filters and
X-ray films (Kodak BiomaxTM MR) were exposed
at –80°C between 1 and 4 days.
Northern Blots
The transfer of RNA from agarose gels on
nylon membranes was performed according to
Chomzcynski (1991). Membranes (also from slot
blot assays) were baked at 120°C for 30 min. The
blots were prehybridized with 6 × SSC, 0.1 % SDS,
2 × Denhardt’s reagent, and 0.01 % DNA from
Both actin and tubulin concentrations change
with time even in controls (Fig. 2A). This is more
pronounced for tubulin, which increases from 1.6
to 4.0% of the total cell protein within one week.
The corresponding values for actin are 1.3 to 1.9%.
The percentage of actin and tubulin is in the range
that is described for other non-muscular cells (Kreis
Fig. 2. Changes of actin (triangles) and tubulin (squares)
concentrations with age (A) and after hormone treatment
(B). In B the influence of 1 µM 20-OH-ecdysone on the
amount of actin and tubulin is demonstrated. At each time
point, the values for the control are set as 100%. In an
ecdysteroid-resistant cell line from Chironomus tentans
(---) there was no change in tubulin concentration as compared to the control.
RESULTS AND DISCUSSION
Actin and Tubulin in Chironomus tentans
and Vale, 1993). After addition of 1 µM 20-OH-ecdysone, there is a transient increase in both cytoskeletal
proteins as compared to controls leading to normal
or even lower values after 6 days (Fig. 2B). The
effect of the molting hormone is more pronounced
for tubulin. In an ecdysteroid-resistant cell line
(Spindler-Barth and Spindler, 1998), there is no
change in actin (Fig. 3) and tubulin concentration
(Fig. 2B) as compared to controls. Molting hormone
does not change the degree of actin polymerisation
(Fig. 3), which is in contrast to D. melanogaster cells,
where in addition to an increase in actin (Couderc
et al., 1982, 1987) and tubulin synthesis (Montpied
et al., 1988) the percentage of fibrillar actin also
increases (Couderc et al., 1982).
Since no C. tentans actin and tubulin cDNAs
are characterized so far, degenerate oligonucleotides were designed based on highly conserved
regions in actin (amino acids 78–87 for the sense
primer, and 222–230 for the antisense primer)
and tubulin (amino acids 264–273 for the sense
Fig. 3. Influence of 1 µM 20-OH-ecdysone on the percentage of G- and F-actin in wild type cells and an ecdysteroid
resistant cell line from Chironomus tentans. Black bars represent G-actin, stippled bars F-actin. Total actin concentration increased in wild type cells after hormone treatment,
but the degree of polymerization remained constant. The resistant clone did not respond to hormone.
15
primer, and 402–411 for the antisense primer).
With these primers, cDNA gained from poly A+RNA was amplified. PCR products of the expected
length (452 bp for actin, 433 bp for tubulin) were
generated and directly sequenced. As expected,
there were some other fragments in addition to a
main fragment, allowing an accurate determination of the sequence only in the core region. This
might be due to the existence of actin and tubulin isotypes and is supported by the finding that
the digoxigenin labelled 452 bp actin probe hybridized to three chromosomal loci and the 433
bp tubulin probe to three separate loci (Fretz et
al., 1998). The core region shows a sequence identity at the amino acid level of 98% to D. melanogaster and 96% to mouse for actin, and 87% for
tubulin for both species (Table 1). Despite this
high sequence similarity, heterologous probes
from mouse were unable to detect actin and tubulin mRNA from C. tentans and vice versa under our hybridization conditions. This might be
due to the fact that C. tentans has a higher AUcontent than D. melanogaster or mouse as calculated from the codon-usage table (Wada et al.,
1992). The corresponding values are 55% for C.
tentans (5.807 codons from 36 mRNAs), 47% for
mouse (4.900.000 codons from 1294 mRNAs),
and 45% for D. melanogaster (249.748 codons
from 550 mRNAs).
With these homologous probes, mRNA for
actin and tubulin were quantified in relation to hormonal treatment. The accuracy of the determination of RNA concentration and of the blotting
efficiency was checked by staining the blots with
methylene blue and subsequent quantitation. The
deviations from the mean were 9.7% (n = 10). Both
probes gave only one signal in Northern blots at
about 1.9 kb (actin) and 2.1 kb (tubulin). Comparable to the profiles for actin and tubulin (Fig. 2),
the corresponding mRNAs also changed after addition of moultmolting hormone (Fig. 4). The increase
in protein and mRNA concentration is similar both
for actin (about 50%) and tubulin (100–110%). This
is suggestive for a transcriptional control of actin
and tubulin under the influence of 20-hydroxyecdysone, although an influence of the hormone on
mRNA stability cannot be excluded.
The interrelationships between the molting
hormone system and cytoskeletal proteins are
rather complex. For example, there is a coregula-
16
Fretz and Spindler
TABLE 1. Comparision of the Core Regions of Actin and b-Tubulin From the Chironomus tentans Cell Line
With Drosophila melanogaster and Mouse*
*Only differences to the Chironomus sequence are marked. For comparison, mouse mRNA for cytoplasmic β-actin (pAL 41;
AA 27-375) and β-tubulin (isotype Mβ 5; AA 1-449) and D. melanogaster Act 87 E gene for actin, and 60 C for β-tubulin
gene were used.
tion of ecdysteroid and β tubulin synthesis in the
prothoracic glands of Manduca sexta where prothoracicotropic hormone induces β tubulin expression (Rybczynski and Gilbert, 1995). In wing discs
of Bombyx mori, mRNA coding for α tubulin is accumulated and reaches maximum values when the
ecdysteroid titre is highest at the end of the fifth
larval instar. Nevertheless, there is no direct influence of ecdysteroids on α tubulin expression, since
in intermolt periods of the fourth and fifth larval
instar when no molting hormone is detectable in
hemolymph, the mRNA titre for α tubulin is also
high, and in addition there is no increase in α tubulin mRNA in wing imaginal discs in vitro after
addition of 20-OH-ecdysone (Hachouf-Gherras et al.,
1998). The situation described here for the C.
tentans cell line is similar to D. melanogaster Kccells: despite the different morphogenetic responses
to ecdysteroids in D. melanogaster (single cells, elongation of the cells with an axon-like outgrowth) and
Fig. 4. Northern blot analysis: 10 µg total RNA from
Chironomus tentans cells at different times of treatment with
1 µM 20-OH-ecdysone were separated electrophoretically,
blotted on Nylon membrane, and hybridized with 32P-labelled
Chironomus specific actin, and tubulin probes. RNA from
control cells was treated the same way.
Actin and Tubulin in Chironomus tentans
C. tentans (multicellular vesicles, change in cell
shape and cell arrangement), actin and tubulin
seem to be influenced in the same way. There is an
increase in actin concentration (Couderc et al., 1982)
and in actin mRNA (Couderc et al., 1987) and the
same is also true for tubulin (Sobrier et al., 1986,
1989; Montpied et al., 1988), but, in contrast to D.
melanogaster, the relation between globular and
fibrillar actin does not change in C. tentans cells.
Interestingly, in wing imaginal discs from the silkworm B. mori actin, mRNA also increases about 2to 3-fold after addition of molting hormone, whereas
in larval silk glands the amount of actin mRNA
drastically decreases (Abraham et al., 1993; Mounier and Prudhomme, 1991). This similar response
between imaginal discs and the C. tentans cell line,
and also the degree of the biological response to
molting hormone, is a further argument that this
cell line is probably of imaginal disc cell origin, as
already noticed for both morphological and other
biochemical parameters (Spindler-Barth and Spindler, in press).
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