Immunofluorescence Localization of a Small Heat Shock Protein (hsp 23) in Salivary Gland Cells of <i>Drosophila melanogaster</i>

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Immunofluorescence Localization of a Small Heat Shock Protein (hsp 23) in Salivary Gland Cells of Drosophila melanogaster

ARRIGO, André-Patrick, AHMAD-ZADEH, C.

Abstract

An aggregate present in cell-free extracts of Drosophila melanogaster tissue culture cells, sedimenting at 20 to 30S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluorescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25° C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambignously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25° C, they move to the cytoplasm.

ARRIGO, André-Patrick, AHMAD-ZADEH, C. Immunofluorescence Localization of a Small Heat Shock Protein (hsp 23) in Salivary Gland Cells of Drosophila melanogaster . Molecular and General Genetics , 1981, vol. 184, no. 1, p. 73-79

DOI : 10.1007/BF00271198

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Mol Gen Genet (1981) 184:73-79

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Immunofluorescence Localization of a Small Heat Shock Protein (hsp 23) in Salivary Gland Cells of Drosophila melanogaster

A.-P. Arrigo i and C. Ahmad-Zadeh z

1 D@artement de Biologic mol6culaire, Universit6 de Gen~ve, 30, quai Ernest-Ansermet, CH-1211 Gen6ve 4, Suisse z Institut d'Hygi6ne, 22, quai Ernest-Ansermet, CH-1211 Gen6ve 4, Suisse

Summary. An aggregate present in cell-free extracts of

Drosophila melanogaster

tissue culture cells, sedimenting at 20 to 30 S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluo- rescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25 ° C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambig- uously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25 ° C, they move to the cytoplasm.

Introduction

Heat shock of

Drosophila melanogaster

brings about the activa- tion of a small number of specific genes (Ritossa 1962; 1964) leading to the synthesis of several new polypeptide chains, the heat shock proteins (Tissi6res et al. 1974; Lewis etal. 1975;

McKenzie et al. 1975). These proteins are characterized by molecular weights of 22, 23, 26, 27, 68, 70 and 84 thousand daltons and are abbreviated here hsp 22, 23, etc. The novel preferential synthesis of particular polypeptides after heat shock appears to be a general phenomenon, as it has been observed in a variety of different organisms from yeast to man (Kelly and Schlessinger 1978; Miller et al. 1979; Voellmy, Bromley and Arrigo, unpub- lished observations).

The function of these proteins is so far unknown. As an approach to this problem, it is important to know their location in the cell. It has been shown recently, by fractionating the cell components after lysis, that following heat shock of tissue culture cells of

Drosophila melanogaster

over 80% of the small heat shock proteins hsp 22, 23, 26 and 27 are found in the nucIei associated with chromatin, while on further incubation at 25°C they move to the cytoplasm. In cytoplasmic extracts they appear to be present in aggregates sedimenting at 20-30S (Arrigo et al. 1980). In the work presented here, these aggregates were isolated from cytoplasmic extracts of tissue culture cells, some of their properties were investigated and hsp 23, 26 and 27 were purified from them. Each of these three proteins was injected into one of three different rabbits. A specific hsp 23 antiserum was obtained. In our experiments injection of hsp 26

Abbreviations:

NP-40: Nonidet P40. PMSF: Phenylmethylsulfonyl- fluoride. SDS: Sodium dodecyl sulfate. EDTA: Ethylene diamine te- traacetic acid. TCA: Trichloroacetic acid. hsp 22, hsp 23 etc.: heat shock proteins of 22,000, 23,000 daltons etc. molecular weight

Offprint requests to:

A.-P. Arrigo

or 27 did not lead to the production of anti-hsp 26 or 27 antibodies.

By means of the antibody raised against hsp 23, immunofluorescence observation of salivary gland cells indicated that after heat shock most of hsp 23 was found in nuclei, while upon incubation of the cells at 25 ° C after heat shock, it was seen to move to the cytoplasm. This observation clearly confirmed;

for hsp 23, the conclusion drawn with isolated nuclei that after heat shock all heat shock proteins, with the exception of hsp 84, were found in nuclei (Mitchell and Lipps 1975; Arrigo et al.

1980) and upon return to 25°C after heat shock they move to the cytoplasm (Arrigo et al. 1980). Our results are in agree- ment with recent observations on autoradiographs of cell sections labeled during heat shock (Velasquez et al. 1980).

Materials and Methods

Cell Culture and [35S] Methionine Labeling of Proteins

The

Drosophila melanogaster

tissue culture cell line KC 161, received from Dr. G. Echallier, was adapted to grow in suspension in D22 medium (Echallier and Okanessian 1970) and the cells, concentrated 10 to 50 times in this medium devoid of methionine, were labeled with 100 ~tCi/ml [35S] methionine (New England Nuclear or Amersham, 300-500 Ci/mmole) for one hour at 25 ° C or 37 ° C, as previously described (Arrigo et al. 1980).

Protein Analysis

Proteins were electrophoresed in 12.5% polyacrylamide gels in presence of SDS according to Laemmli (1970) as previously described (Arrigo et al. 1980).

When non-denaturing conditions were used, Ficoll and bro- mophenol blue were added to the proteins eluted from the G-150 Sephadex column to reach a concentration of 10% and 0.01%

respectively. These protein solutions were then electrophoresed at 200 V for 90 rain in slab gels of 0.5% agarose and 2% acrylamide according to Peacock and Dingmann (1968).

Cesium Chloride Gradients

Analysis of aggregates containing native hsp 23, 26 and 27 on CsC1 was carried out as previously described (Arrigo et al. 1980).

Antisera and Imrnunoprecipitation

Rabbits were immunized with purified hsp 23, 26 and 27. 500- 750 ~tg of heat shock proteins, emulsified in complete Freund's

0026-8925/81/0184/0073/$01.40

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Fig. 1 a, b. SDS polyacrylamide gel electrophoretic analysis of fractions from sucrose gradients.

a Coomassie stained gels. b Autoradiography. Track T represents total cells lysate, RP ribosomal pellet and B fraction at the bottom of the gradient

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Fig. 2A, B. Analysis of the partially purified proteins. Following chromatography in G-t50 Sephadex, the aggregates containing the heat shock proteins were analysed on SDS polyacrylamide gels as described in Materials and Methods. A Coomassie blue stained gels. B Autoradi- ography. Track a represents whole cells, track b represents partially purified proteins

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Fig. 3a-c. Cesium chloride analysis of the aggregate. The aggregate containing the small heat shock proteins was analysed in CsCI as described in Materials and Methods. a Aggregate without prefixation in formaldehyde, b Aggregate after fixation with formaldehyde, e The aggregate was treated for 1 h at 20 ° C with 20 gg/ml of pancreatic RNase before prefixation with formaldehyde

adjuvant, were given in two injections, 4 weeks apart. The sera were collected 3 weeks after the last injection.

For immunoprecipitation, 35S methionine labeled cells were lysed in a solution containing 50 mM tris pH 7.4, 5 mM EDTA, 150 mM NaC1, 0.5% NP-40, 1 M Urea and PMSF. After centrifugation at 10,000 g for 10 min aliquots of tysate corresponding to 10 30 x 106 cells were incubated with 15 gl of either immune or control sera and 30 gl of protein A-Sepharose (Pharmacia) overnight at +4 ° C. After washing with lysis buffer, proteins were eluted from protein A-Sepharose columns and analysed on SDS-PAGE.

Cellular Localization of hsp Protein by Immunofluorescence

The salivary glands from third instar larvae, heat shocked or kept at 25 ° C, were dissected and dipped into 2% TCA for 5 rain. Two or three glands were then placed on a microscope

slide and squashed by means of a siliconized cover slip. After this operation, the cells should remain individually intact and be well dispersed on the slide. The slides were then frozen on' dry ice and the cover slip was removed with forceps. The preparation was fixed by dipping in acetone for 15 rain. The slides, after drying, could be kept for several weeks at -20 ° C. To the slides, 100 gl of the appropriate dilution of the antiserum was added and after incubation for 30 rain in a small damp chamber, the slides were rinsed three times for 5 rain each in a solution containing 10 mM tris pH 7.4 and 150 mM NaC1.

The antisera were usually diluted 2-40-fold in this solution in the presence of 1 mg/ml bovine serum albumin. The second incubation with antibodies was made as described above with anti- rabbit fluorescent goat serum (Behring Institute) diluted 50 times in tris buffer-saline (see above). This operation took place in the dark. After 5 washes in tris buffer-saline, a drop of a solution containing 90% glycerol and 10 mM tris-HCl, pH 8.1, was added to the preparations. A non-siliconized cover-slip was then'(tepos-

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ited on each preparation. The preparations were examined with a phase contrast microscope fitted with a fluorescence attach- ment. The photographs were made on Kodak Tri-X400 ASA films.

Fig. 4a-c. Analysis of the specificity of the anti-hsp 23 serum. The specificity of the anti-hsp 23 serum was analysed using the protein A-sepharose method (Maln6e et al. 1979) in presence of 1 M urea to dissolve the low molecular weight heat shock proteins. As antigen, total cell lysate in 1 M urea was used. The cells had been labeled for 1 h at 37 ° C with [3SS]methionine before the lysis. Immunoprecipi- tated proteins were analysed on 12.5% SDS polyacrylamide gels, as described in Materials and Methods. The autoradiograph of the gels is shown, a Control showing the position of the heat shock proteins in a total cell lysate, b Specificity of the preimmune serum, c Specificity of the anti-hsp 23 serum

Results

a) Purification of hsp 23, 26 and 27

To purify these proteins, advantage was taken of the following observation. When the cells are brought back to 25°C after a 1 h heat shock, the small heat shock proteins, mainly present in the nucleus bound to chromatin at 37 ° C, move to the cytoplasm (Arrigo et al. 1980). In a cell lysate hsp 23, 26 and 27 are then present in an aggregate sedimenting at 20-30S resembling RNPs (Preobrazhensky and Spirin 1978). Hsp 22, which is present in the total cytoplasmic extract, is not recovered in the aggregate (Fig. 1). Thus the first step in the purification of hsp 23, 26 and 27 was the isolation of the aggregate. About 101° cells were incubated for 1 h at 37 ° C in 20 ml of Dz2 medium devoid of methionine to which 50 gCi/ml of 3ss methionine had been added. They were brought back to 25 ° C and further incubated at this temperature for 6 h. They were then lysed in a solution containing 10 mM triethanolamine pH 7.4, 10 mM NaC1, 5 mM MgC12, 0.1% Triton X-100 and 1 mM/3-mercapto- ethanol and the lysate was centrifuged for 10 min at 16,000 g.

The supernatant was centrifuged for 3 h at 150,000 g. The pellet formed mainly of ribosomes and of the aggregate was resus- pended in lysis solution and centrifuged in sucrose gradients (0.5-1.5 M sucrose, 10 mM triethanolamine pH 7.4, 10 mM NaC1 and 5 mM MgC12) for 13.5 h at 40,000 rpm. in an SW40 rotor on a Spinco centrifuge. From each fraction, collected from the bottom of the tube, an aliquot was precipitated at 4°C with TCA and analyzed by electrophoresis on SDS polyacrylamide gels. The results are shown in Fig. 1. In the gradient, 80S ribosomes and polysomes were pelleted (fraction B) while the aggregate containing hsp 23, 26 and 27 sedimented at 20 to 30S and was recovered in fractions 5 8. The same aggregates could also be observed by analysing the cell lysate directly in sucrose gradients, thus avoiding the resuspension of the aggregates pelleted by high speed centrifugation.

In the same type of experiment, when the 6 h incubation at 25°C after heat shock was omitted, no labeled hsp 23, 26 and 27 were recovered from the gradient. This was due to the fact that, under these conditions, these proteins remained associated with chromatin (Arrigo et al. 1980). It is interesting that when cells were kept at 37 ° C, or when they were not heat shocked and thus were kept at 25 ° C, protein bands corresponding to hsp 23, 26 and 27 were found weakly stained with coomassie blue. This would indicate the presence of a small and rather stable cytoplasmic pool of these proteins under normal conditions.

Fig. 5. Cellular localization of hsp 23 by immunofluorescence in salivary glands cells. 1. A salivary gland cell seen in phase contrast from a larva heat shocked for 1 h at 37 ° C and immediately dissected, and squashed x 250. 1'. As 1. but indirect immnnofluorescence of preimmune serum with fluorescent goat anti-rabbit serum. 2. As 1. but kept at 25 ° C, x500. 2'. As 2. but indirect immunofluorescence of anti-hsp 23 serum with fluorescent goat anti-rabbit serum. 3. Cell in phase contrast from a larvae heat shocked for 1 h at 37 ° C and immediately squashed, x 500. Y. As 3. but indirect immunofluorescence of anti hsp-23 serum with fluorescent goat anti-rabbit serum. 4. Cell in phase contrast from a larvae heat shocked for 1 h at 37 ° C and kept 3 h at 25 ° C before being dissected. 4'. As 4. but indirect immunofluorescence of anti-hsp 23 serum with fluorescent goat anti-rabbit serum

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Fractions 5-8 from the gradient (Fig. 1) which were enriched in hsp 23, 26 and 27 were pooled, the NaC1 concentration brought to 250 mM and the mixture loaded on a G-150 Sephadex column. The proteins were eluted with a solution containing 10raM triethanolamine pH 7.4, 5mM MgC12 and 250mM NaC1. The proteins present in the void volume which contained hsp 23, 26 and 27 were precipitated in the cold with TCA and electrophoresed on 12.5% polyacrylamide SDS gels. The results are shown in Fig. 2. It is seen that some of the high molecular weight proteins were removed by this step, though some unla- beled proteins copurified with the heat shock proteins (Fig. 2, Ab). Hsp 23, 26 and 27 were eluted from the gels by electrophoresis as described by Mirault et al. (1978). The proteins were then chromatographed on a column of G-25 Sephadex and eluted with twice-distilled water. In this last step most of the SDS was eliminated.

b) Some Properties of the Aggregate Containing hsp 23, 26 and 27

The absorption spectrum of the aggregate after the G-150 Sepha- dex chromatography showed a main peak at 260 nm, suggesting the presence of nucleic acids and a shoulder at 280 nm. This was confirmed by the analysis of the composition of the aggregate in protein, RNA and DNA by the methods of Lowry (1951), orcinol (Ashwell 1957) and diphenylamine (Burton 1956) respectively. The ratio of 50:9:1 was found for proteins RNA and DNA respectively. The analysis of the aggregate in CsC1 density gradients is shown in Fig. 3. Without prefixation with formaldehyde, the proteins labeled with [35S] methionine banded at a density of 1.31 g/ml, corresponding to the density of free proteins. After formaldehyde fixation the density was 1.36 g/ml with a shoulder at 1.42 g/ml. Pancreatic RNase (20 gg/ml for 1 h at 20 ° C) preceding the fixation with formaldehyde suppressed the shoulder at 1.42 g/ml, without affecting the main peak. Tak- ing into account the ratio of protein, RNA and DNA given above, it can be concluded that the main peak should still contains RNA after RNase treatment. Thus this RNA would not be accessible to RNase. In non-denaturing gels (2% acrylamide and 0.5% agarose according to Peacock and Dingman (1968);

see Materials and Methods), the aggregate was not dissociated and migrated as one large diffuse band. When the proteins in this band were electrophoresed in a second dimension in SDS, hsp 23, 26 and 27 were clearly seen.

The preliminary results described above suggest that the aggregate containing hsp 23, 26 and 27 shares at least some of the properties of RNPs (Preobrazehnsky and Spirin 1978).

c) Specificity of Rabbit Antisera to hsp 23, 26 and 27

The specificity of antisera was tested by reacting with soluble extracts of labeled cells and analysis of solubilized immune com- plexes on SDS-PAGE as described in Materials and Methods.

Figure 4 shows that hsp 23 was selectively precipitated by anti- hsp 23 serum from lysate of heat shocked culture cells (Fig. 4c).

This polypeptide was absent when control serum was used (Fig. 4b). While anti-hsp 23 serum reacted with hsp 23 (lane c), two faint bands, at the position of hsp 84 and 70 were present with control serum (lane b) and also with the anti-hsp 23 serum (lane c). This effect does not appear to be specific. Anti-sera prepared against hsp 26 and 27 did not contain any detectable antibodies to heat shock proteins by the technique used.

d) Localization of the Low Molecular Weight Heat Shock Proteins in Salivary Glands Cells

by Indirect Immunofluorescence

Third instar larvae of

Drosophila melanogaster,

strain Kolmar, were heat shocked for 1 h at 37 ° C. The salivary glands were dissected immediately after the shock or after a period of 3 h at 25°C after the shock. Control glands were isolated from larvae kept at 25 ° C. The salivary glands were squashed, the cells fixed with acetone and the low molecular weight heat shock proteins were localized by indirect immunofluorescence (see Ma- terials and Methods). A strong fluorescence localized in the nuclei only was seen in salivary gland cells of heat shocked larvae incubated with anti hsp 23 serum (Fig. 5). No fluorescence was seen in the cells of salivary glands from heat shocked larvae incubated with preimmune serum. The same negative result was obtained when salivary gland cells from larvae kept at 25°C were incubated with anti hsp 23 serum. However in this case only a weak fluorescence was seen in the cytoplasm. This might be due to a small pool of low molecular weight heat shock proteins present normally at 25 ° C, as was suggested above by the presence of weak Coomassie stained bands in SDS polyacrylamide gels.

The results indicate that in the salivary gland cells from heat shocked larvae, most of the small heat shock proteins are localized in the nuclei. In salivary gland cells from larvae kept 3 h at 25 ° C after a heat shock, a strong fluorescence was seen in the cytoplasm, while the fluorescence in the nuclei, though still present, was clearly weaker than was the case directly following the shock.

Discussion

By means of immunofluorescence observation of salivary gland cells, we find that the majority of the small heat shock protein hsp 23 is present in the nuclei following a heat shock, and moves to the cytoplasm on further incubation at 25 ° C. This confirms, for hsp 23, earlier reports made with preparations of salivary gland cells (Mitchell and Lipps 1975) and tissue culture cells (Arrigo et al. 1980). It is thus a natural hypothesis that the site of action of hsp 22, 23, 26 and 27 is in the nucleus, probably in chromatin (Arrigo et al. 1980 ; Arrigo 1980 ; Velasquez et al.

1980).

In cytoplasmic extracts, hsp 23, 26 and 27 sediment at 20 to 30S in aggregates having a composition resembling RNP (Pre- obrazhensky and Spirin 1978).

In the present work, injection of purified hsp 23 into a rabbit led to the production of anti-hsp 23 antibodies~ The antiserum against hps 23 thus obtained precipitated hsp 23 but not hsp 22, 26 and 27. This is not surprising as the four small heat shock proteins all showed distinct tryptic fingerprints (Mirault et al.

1978) and no cross-hybridization was observed between the small heat shock genes (R. Voellmy, personnal communication).

Acknowledgments.

This work was supported by a grant from the Swiss National Science Foundation No. 3.512.79 to A. Tissi6res. We thank O. Jenni and Y. Epprecht for drawings and plates, C. Tonka for the cell cultures, B. Brun for typing the manuscript, M.-E. Mirault, P.-F. Spahr and R. Jackson for helpful discussions, and M.-E. Mirault for his help in the performance of some experiments, We are particular- ly indebted to A. Tissi+res for his advice and encouragement through- out this work.

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