Hormonal regulation of actin and tubulin in an epithelial cell line from Chironomus tentans.код для вставкиСкачать
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). LITERATURE CITED Abraham EG, Mounier N, Bosquet G. 1993. 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