Ultrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods

Journal of Structural Biology 166 (2009) 329–336

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Ultrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods Christine Charlier, Françoise Lamaye, Nicolas Thelen, Marc Thiry * Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, 20, rue de Pitteurs, 4020 Liège, Belgium

a r t i c l e

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Article history: Received 10 December 2008 Received in revised form 27 February 2009 Accepted 3 March 2009 Available online 12 March 2009 Keywords: Cell nucleus RNA DNA Cytochemistry Immunocytochemistry

a b s t r a c t The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock has been shown to increase their number, we applied a hyperthermal shock on HeLa cells to investigate the nucleic acid content of PGs by means of cytochemical and immunocytological approaches. These heat shock-induced PGs (hsiPGs) appeared as clusters organized in the form of honeycomb structures and were always associated with some blocks of condensed chromatin, such as the perinucleolar chromatin shell. A stalk connecting the hsiPG to the chromatin could be observed. For the detection of RNA, we applied an immunocytological method involving two anti-RNA antibodies and quantified the gold labelling obtained. The results clearly revealed that hsiPGs contained RNA. Regarding to the detection of DNA, we used three different methods followed by quantitative analyses. The results seemed to indicate that a small amount of DNA was present in hsiPGs. Together, these findings suggest that hsiPGs might be RNP structures associated with particular regions of DNA. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Like the cytoplasm, the nucleus contains various structurally and functionally different subcompartments (Fakan and Van Driel, 2007). The best known is the nucleolus, which is mainly involved in the ribosome biogenesis (Raska et al., 2006). By contrast, other structures as the perichromatin granules (PGs) remain very enigmatic at the present time, although they have been described for a long time (Watson, 1962). The PGs are spherical structures of approximately 40–55 nm in diameter, surrounded by a clear halo of about 20–25 nm (Watson, 1962; Monneron and Bernhard, 1969; Vazquez-Nin and Bernhard, 1971; Daskal, 1981; Puvion and Moyne, 1981). PGs appear only seldom as clustered complexes and are most frequently found at the surface of condensed chromatin. Sometimes, they are linked to the latter by a thin filament (Monneron and Bernhard, 1969; Daskal, 1981). The number of PGs is estimated at between 500 and 2000 by nucleus of mammalian cells (Watson, 1962; Monneron and Bernhard, 1969) and varies depending on the physiological conditions. For examples, an increased number of PGs has been observed when the cells are subjected to hyper- or hypo-thermal shocks (Heine et al., 1971; Puvion et al., 1977; Cervera and Montero, 1980; Mähl et al., 1989) and when the cells come into apoptosis (Miller et al., 2002) or hibernation (Biggiogera and Pellicciari, * Corresponding author. Fax: +32 4 366 51 73. E-mail address: [email protected] (M. Thiry). 1047-8477/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2009.03.002

2000). Cycloheximide, an inhibitor of protein synthesis (Daskal et al., 1975; Lafarga et al., 1993), a-amanitin, an inhibitor of polymerase II and III (Derenzini and Moyne, 1978), 5-6-dichloro-1-bribofuranosyl benzimidazole, an inhibitor of mRNAs synthesis (Puvion et al., 1981), all cause an increase in the number of PGs in treated cells. By contrast, a decrease of PGs has been reported during the autolysis of normal cells (Karasek, 1975), during the differentiation of erythroblasts (Zs-Nagy et al., 1977), during aging (Zs-Nagy et al., 1977), at the awakening of hibernating dormouse (Zancanaro et al., 1993) and with dehydration (Lafarga et al., 1993). PGs are visible throughout the cell cycle. During mitosis, they meet preferentially around the chromosomes (Puvion and Moyne, 1981). Moreover, they are not only found in animals (Romanova, 1978; Locke and Huie, 1980; Raikova, 1980; Volonterio and Ponce de Leon, 2004) but also among plants (Jiménez-Ramírez et al., 2002) and protists (Esponda et al., 1983; Alverca et al., 2006). Some cytochemical studies were devoted to the PGs. These were almost all qualitative investigations. Chemical and enzymatic extractions have supported the view that PGs are RNP structures (Monneron and Bernhard, 1969; Vazquez-Nin and Bernhard, 1971; Smetana et al., 1979). Similarly, two cytochemical techniques, the EDTA regressive staining procedure (Monneron and Bernhard, 1969) and the terbium staining (Biggiogera and Fakan, 1998), have confirmed the RNP nature of PGs. A presence of RNA in PGs was also detected through the RNase-gold complex (Cheniclet and Bendayan, 1990). Two methods of EM in situ hybridization have further shown that PGs could contain polyade-

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nyled RNA (Visa et al., 1993a) and the snRNAs U1 and U2 (Visa et al., 1993b). Finally, immunocytochemical methods have revealed the presence of proteins associated with pre-mRNAs and snRNAs in PGs (Fakan et al., 1984; Puvion et al., 1984; Vazquez-Nin et al., 1994; Malatesta et al., 1999; Chiodi et al., 2000). As to the presence of DNA, current data are controversial. Digestions with DNase alone (Vazquez-Nin and Bernhard, 1971) or combined with pepsin (Smetana et al., 1979) applied on rat hepatocytes could not attest the presence or absence of DNA in PGs. After more specific DNA staining techniques such as thallium ethylate (Moyne, 1973) and osmium-ammine (Moyne et al., 1977), no PGs were revealed. However, Si (1993) detected DNA of human papillomavirus type 11 in PGs of human uterine cervical carcinoma cells by EM in situ hybridization. Until now, the function of PGs remains unknown. The most often advanced hypothesis considers PGs as structures playing a role in the transport of mRNA within the nucleus and/or in its intranuclear storage (Monneron and Bernhard, 1969; Puvion and Moyne, 1981; Fakan et al., 1984; Takeuchi and Takeuchi, 1988; Vazquez-Nin et al., 1996). This view is essentially based on the fact that the mammalian PGs have many points in common, both in morphology and composition, with Balbiani granules which are nuclear mRNP particles of Drosophila and Chironomus (Daneholt, 2001). However, unlike the Balbiani granules, any passages of PGs through nuclear pores have not been viewed so far. In the present study, we have re-evaluated the nucleic acid content of PGs in HeLa cells. For this, we submitted our cells to a hyperthermal shock, treatment known to increase the number of PGs in the nucleus of mammalian cells (Cervera and Montero, 1980; Mähl et al., 1989). Under these experimental conditions, we applied different cytochemical and immunocytological approaches to detect RNA and DNA at the ultrastructural level. Our results seem to indicate that hsiPGs contain both nucleic acids. 2. Materials and methods 2.1. Biological materials HeLa cells were grown at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium (Gibco-BRL, Life Technologies, Gent, Belgium) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 100 U/ml penicillin. Some cell cultures were submitted to a heat shock (42 °C for 1 h) before to be reincubated at 37 °C during 45 min to 4 h. A part of these cell cultures was incubated for 19 h in medium containing bromodeoxyuridine (BrdU, 5  10 6 M, Boehringer Mannheim) and 5-fluoro-2-deoxyuridine (FdU, 5  10 7 M, Sigma). 2.2. Electron microscopy Cell pellets were fixed for 40–60 min at 4 °C in 4% formaldehyde (Ladd Research Industry, Burlington, Vermont, USA) or 1.6% glutaraldehyde (Ladd) or 4% formaldehyde/0.15% glutaraldehyde in 0.1 M Sörensen’s buffer (pH 7.4). They were dehydrated through graded ethanol or acetone solutions, and embedded in Epon or in Lowicryl K4M as in Roth et al. (1981). Some 60 min 1.6% glutaraldehyde-fixed samples were acetylated according to Wassef et al. (1979) in order to obtain a good distinction of various nuclear components. Ultrathin sections were either collected in platinum rings (4 mm diameter) formed by a platinum wire (0.1 mm diameter, SA Johnson Matthey, Brussels, Belgium) and stored in distilled water until used or mounted on colloidoncoated grids. Finally, ultrathin sections were stained with uranyl acetate and lead citrate before examination in a Jeol CX 100 II electron microscope at 60 kV.

2.3. Cytochemical methods Bernhard’s EDTA regressive staining (Bernhard, 1968). Thin sections of acetylated cells were incubated for 5 min at room temperature in darkness, on drops of 50% ethanolic uranyl acetate, rinsed in three 25 ml-beaker filled with boiled deionised water, floated on drops of 0.2 M EDTA (pH 7) for 10–60 min, transferred on drops of aqueous lead citrate for 5 min, rinsed in three 25 ml-beaker filled with boiled deionised water and dried on filter paper. Feulgen-like osmium-ammine staining for detecting DNA (Cogliati and Gautier, 1973; Olins et al., 1989). Sections of formaldehyde-fixed and Lowicryl K4M-embedded cells were mounted on gold grids. The latter were floated on 3.5 N HCl for 30 min at 37 °C, rinsed with deionised water and then reacted with 0.1% osmium-ammine solution (treated with SO2 for 30 min) for 40 min at 37 °C in darkness. Sections were then rinsed with deionised water and dried. Two control experiments were carried out: no staining was observed when grids were either stained with osmium-ammine solution without prior hydrolysis or when floated on osmium-ammine solution without prior bubbling with SO2. 2.4. Immunocytological methods In situ Terminal deoxynucleotidyl transferase (TdT)-Immunogold labelling method for detecting DNA (Thiry, 1992). Grids containing thin sections of acetylated cells were incubated for 15 min at 37 °C at the surface of the following medium (pH 6.5): 20 lM 5 bromo-2-deoxyuridine (BrdU) triphosphate (Sigma, St. Louis, MO, USA), 100 mM sodium cacodylate (pH 7.2), 2 mM MnCl2, 10 mM b-mercaptoethanol, 50 lM/ml BSA, 125 U/ml calf thymus TdT (Boehringer Mannheim; Mannheim, Germany) and 4 lM each of dCTP, dGTP and dATP (Gibco-BRL; Merelbeekee, Belgium). Then, the sections were rinsed twice in double-distilled water and incubated for 20 min at room temperature in PBS (0.14 M NaCl, 6 mM Na2HPO4, pH 7.2) containing normal goat serum (NGS) diluted 1:30 and 1% BSA, then rinsed with PBS containing 0.2% BSA. Subsequently, the sections were incubated for 4 h at room temperature with monoclonal antiBrdU antibody (Becton Dickinson; Mountain View, CA) diluted 1:50 in PBS containing 0.2% BSA and NGS diluted 1:50. After washing with PBS containing 1% BSA, the sections were incubated at room temperature for 1 h with goat anti-mouse IgG coupled to colloidal gold (5 nm diameter; Amersham Life Science, Little Chalfont, United Kingdom) diluted 1:40 in PBS containing 0.2% BSA, pH 8.2. After washing with PBS containing 1% BSA, the sections were rinsed with deionised water and dried before to be contrasted. Two control experiments were carried out: no labelling occurred either when the primary antibodies were omitted, or when the sections were incubated with the TdT medium devoid of TdT or labelled nucleotides. Immunocytological method for detecting DNA after in vitro BrdU incorporation (Thiry and Dombrowicz, 1988). Ultrathin sections of formaldehyde/glutaradehyde mixturefixed and Lowicryl K4M-embedded cells were either heated at 100 °C for 5 min and then immediately transferred in deionised water at 4 °C or incubated in 3.5 N HCl for 30 min and then rinsing several times with deionised water. Subsequently, the BrdU-labelled sites were detected using the monoclonal anti-BrdU antibody as described above for the TdT method. The labelling procedure was applied to both faces of ultrathin sections. Two control experiments were carried out: no labelling was observed either when the primary antibodies were omitted, or when the immunocytological method was applied on sections of cells that are not exposed to BrdU and FdU.

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2.5. Immunocytological method for detecting RNA For the immunocytological localization of RNA two mouse monoclonal anti-RNA antibodies (D444, BWR5) were essentially applied as previously described (Thiry, 1992). Ultrathin sections of 1.6% glutaradehyde-fixed and Lowicryl K4M-embedded cells were incubated for 20 min in PSBS (34 mM NaCl, 0.7 mM KCl, 20 mM Na2HPO4, 0.4 mM KH2PO4, 1% BSA, pH 7.2) containing normal goat serum (NGS) and normal rabbit serum (NRS), both diluted 1:30. Then, the sections were incubated for 3 h at room temperature in the presence of RNA-specific antibodies diluted 1:10 in PSBS containing NGS and NRS, both diluted 1:50. After five rinses in PSBS, the sections were incubated for 30 min with goat antimouse IgG3 (heavy chain specific; Sigma, St Louis, MO) diluted 1:100 in PSBS containing NSG and NRS, both diluted 1:50. Subsequently, the sections were rinsed four times in PSBS (pH 7.2), plus one in PSBS (pH 8.2) and then incubated for 1 h at room temperature in a medium containing rabbit-goat IgG coupled to 5 nm colloidal gold particles (Amersham) diluted 1:50 in PSBS (pH 8.2). Sections were rinsed with PSBS and then with distilled water before to be mounted on nickel grids coated with collodion. Subsequently, the labelling procedure was applied to the second face of ultrathin sections. Two control experiments were carried out: no labelling was observed either when the primary antibodies and/or the secondary antibodies were omitted, or when the sections were incubated with antibody-free particles. 2.6. Quantitative evaluations Since the electron-dense marker used in the immunogold technique is particulate, the density of labelling can be quantified. As demonstrated previously, only antigenic sites exposed at the surface of the sections can interact with the antibodies (Bendayan, 1984). Therefore, the labelling density is independent of the section thickness but is directly related to the areas occupied by each of the intracellular compartments. Since differences in observed labelling densities reflect relative differences in the concentration of antigenic sites, only relative comparisons between intensities can be considered. To evaluate the labelling density, the area of each compartment (Sa) was estimated using a morphometrical approach by the pointcounting method (Weibel, 1969). Then, the number of gold particles (Ni) present over each compartment was counted and the labelling density (Ns) calculated (Ns = Ni/Sa). 3. Results 3.1. Accumulation of hsiPGs after a thermal shock After application of the acetylation technique on HeLa cells grown at 37 °C, we have observed a few (1–8) PGs by cell nucleus section. These PGs measure approximately 40–60 nm in diameter and are surrounded by a clear halo about 25 nm thick. Generally, PGs are independent of each other and are associated with blocks of condensed chromatin (Fig. 1a). To increase the PG number by cell nucleus, we submitted our cultures to a supranormal temperature (1 h at 42 °C). After the heat shock, we reincubated our cultures at 37 °C during different time periods, to determine the best conditions to get a maximum of PG by nuclear area. On HeLa cells incubated for 1 h at 42 °C in their culture medium, we have found that the structure of hsiPGs do not change compared to PGs of cells grown at 37 °C. By contrast, their quantity by nuclear area increases very significantly (Table 1). The increase continues even when heat-shocked cells are reincubated at 37 °C, reaching a maximum after 90 min. Later, the nuclear density of

Fig. 1. (a) and (b) Effect of a hyperthermal shock on the hsiPGdistribution. HeLa cells grown at 37 °C (Fig. 1a) or exposed to 42 °C for 60 min and then reincubated for 90 min at 37 °C (Fig. 1b) as revealed with the acetylation method. PGs or hsiPGs appear as granules surrounded by a clear halo and associated with clumps of condensed chromatin (C). They appear as elements dispersed in the nucleus of cells cultured at 37 °C (Fig. 1a), while they form clusters in cells cultured at 37 °C after a thermal shock (Fig. 1b). Arrows indicate PGs or hsiPGs. IG: interchromatin granule cluster. Nu: nucleolus. Bars = 0.2 lm.

Table 1 Number of PGs or hsiPGs per micrometer square of nucleus in HeLa cells under different culture conditions as observed with the acetylation method. The results are the means ± SEM. Thirteen to Sixteen randomly chosen micrographs (n) were analysed. Student’s t test was used to compare the PG density in the nucleus of non-treated cells with that observed in other conditions (*P < 0.1, **P < 0.05, *** P < 0.001). Conditions

Mean values ± standard deviation

n

Without treatment 60 min at 42 °C 60 min at 42 °C + 45 min at 37 °C 60 min at 42 °C + 90 min at 37 °C 60 min at 42 °C + 135 min at 37 °C 60 min at 42 °C + 180 min at 37 °C 60 min at 42 °C + 240 min at 37 °C

0.14 ± 0.05 0.23 ± 0.19** 0.24 ± 0.21** 1.19 ± 0.59*** 0.29 ± 0.23** 0.31 ± 0.17** 0.20 ± 0.15*

16 14 13 16 13 16 13

hsiPGs decreases. hsiPGs appear as clusters more or less important depending on whether the cell cultures are reincubated or not at 37 °C after the thermal shock. Without recovery, hsiPGs are gathered in clusters of maximum 20 units. After recovery ranging from 45 min to 4 h, we have often observed clusters of up to 40 units (Fig. 1b). Sometimes, these clusters contain a fibrillar core (Fig. 2). Clusters of hsiPGs are not located randomly in the cell nu-

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Fig. 2. Structural organization of hsiPG clusters induced by hyperthermal shock. HeLa cells exposed to 42 °C for 60 min and then reincubated for 180 min at 37 °C as revealed with the acetylation method. hsiPGs are grouped in the form of a honeycomb structure where each hsiPG (arrows) occupies the centre of a cell. Sometimes, we can see a thin filament (large arrowheads) that connects the granule to the surrounding condensed chromatin (C). In the centre of the hsiPG cluster, we see a fibrillar core (B). Insert: detail of cells containing hsiPGs. The cell wall is formed by a twisted cordon of condensed chromatin (small arrowheads). Bar = 0.05 lm.

cleus but are always associated with large blocks of condensed chromatin, as such the perinucleolar shell of condensed chromatin. As illustrated in the Fig. 1b, the structural organization of clusters is usually in the form of a honeycomb structure where each hsiPG takes up the centre of a cell and where the cell wall is formed by twisted cordons of condensed chromatin (Fig. 2, insert). Sometimes, a stalk connecting the hsiPG to the chromatin can be seen (Fig. 2). 3.2. Detection of RNA in hsiPGs The presence of RNA within hsiPGs was first investigated using the Bernhard’s EDTA regressive staining; a technique that identifies structures rich in RNPs (Bernhard, 1968). When the EDTA staining is applied on blocks of acetylated and heat-shocked cells, a bleaching of condensed chromatin blocks is observed while nucleoli and hsiPGs remain well contrasted. The hsiPGs are clearly visible and their size is similar to that observed in classically contrasted preparations. However, they do not appear at the centre of a halo, originally demarcated by the condensed chromatin, because it is bleached in this case (Fig. 3a). Sometimes, the stalk connecting the hsiPG to the condensed chromatin can still be seen. After this staining for RNP structures, we used an immunogold labelling procedure involving two anti-RNA antibodies. On heatshocked cell sections, gold particles are visualized on the nucleoli, over the nucleoplasm and over some hsiPGs (26.6%, n = 150). The largest part of the condensed chromatin is completely devoid of labelling (Fig. 3b). To estimate the significance of the hsiPG labelling, we carried out a quantitative study. To do this, the number of gold particles per lm2 over the hsiPG was counted on 19 cell nuclei. Using a Student’s t test, we have shown that the average labelling density over hsiPGs (96.97 ± 80.65) is significant compared to that of condensed chromatin (15.95 ± 7.76) considered in this case as background noise. 3.3. Detection of DNA in hsiPGs To detect the DNA, we first used the TdT method because this is the only one that can be directly applied to our acetylated biolog-

Fig. 3. (a) and (b) Cytochemical and immunocytological detection of RNA within hsiPGs. Detection of RNA within hsiPGs from HeLa cells exposed to 42 °C for 60 min and then reincubated for 180 min (Fig. 3a) or 60 min (Fig. 3b) at 37 °C as revealed with the Bernhard’s EDTA staining (Fig. 3a) and with the immunocytological technique using anti-RNA antibodies (Fig. 3b). Fig. 3a: clumps of condensed chromatin (C) are bleached while the hsiPGs (arrows) remain stained. The thin filament connecting the granule to the surrounding condensed chromatin is also contrasted (arrowheads). Fig. 3b: a labelling is present over the interchromatin spaces (arrowheads) and over some hsiPGs (arrows). CYT: cytoplasm. IG: interchromatin granule cluster. NE: nuclear envelope. Bars = 0.1 lm.

ical material, and it is also a very specific technique. The immunodetection of BrdU, added by TdT to DNA ends at the surface of heatshocked cell sections, has shown that gold particles are mainly located over the condensed chromatin (Fig. 4a). A labelling is also observed over the nucleoplasm and over some hsiPGs (49.7%, n = 400), while the cytoplasm, outside the mitochondria, and the clusters of interchromatin granules are devoid of labelling. It should be noted that to increase the labelling precision of this technique, in addition to having used particles of 5 nm in diameter, we shortened, contrary to the protocol described previously (Thiry, 1992), the incubation time of sections with the aqueous solution containing essentially the TdT, BrdU and nucleotides, to avoid too great extension of DNA at the surface of sections. As for the RNA, we conducted a quantitative analysis on 22 cell nuclei to evaluate the significant value of the hsiPG labelling. Our results indicate that the average density of hsiPG labelling is lower than that of condensed chromatin but it is significant compared to that of cytoplasm considered in the case as background noise (Table 2). In a second step, we used a more precise method detecting the BrdU previously incorporated into DNA during cell replication rather than that added to DNA ends at the section surface. This has been strongly recommended by the fact that hsiPGs are usually surrounded by condensed chromatin which is significantly labelled by DNA detection techniques. By this technique, we can see, as

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Table 3 Number of gold particles per micrometer square over the hsiPG, the condensed chromatin and the cytoplasm in HeLa cells replaced at 37 °C after a thermal shock as revealed with the immunocytological method for detecting BrdU previously incorporated into cells during replication. The results are the means ± SEM. Twenty six randomly chosen micrographs were analysed. Student’s t test was used to compare the nuclear components with the cytoplasm (*P < 0.5, **P < 0.01). Mean values ± standard deviation PG Condensed chromatin Cytoplasma a

Fig. 4. (a) and (b) Immunocytological detection of DNA within hsiPGs. Detection of DNA within hsiPGs from HeLa cells exposed to 42 °C for 60 min and then reincubated for 120 min at 37 °C as revealed with either the in situ TdT-immunogold labelling technique (Fig. 4a) or the immunocytological technique for detecting BrdU previously incorporated into cells during replication (Fig. 4b). In addition to an evident labelling of the condensed chromatin (C), some hsiPGs are labelled (arrows). CYT: cytoplasm. IG: interchromatin granule cluster. Bars = 0.1 lm.

Table 2 Number of gold particles per micrometer square over the hsiPG, the condensed chromatin and the cytoplasm in HeLa cells replaced at 37 °C after a thermal shock as revealed with the in situ TdT-immunogold labelling method. The results are the means ± SEM. Twenty two randomly chosen micrographs were analysed. Student’s t test was used to compare the nuclear components with the cytoplasm (*P < 0.1, ** P < 0.01). Mean values ± standard deviation PG Condensed chromatin Cytoplasma a

483.86 ± 181.00* 655.75 ± 250.70** 1.85 ± 2.30

Mitochondria excepted.

with the previous technique, that condensed chromatin and some hsiPGs (44.5%, n = 400) are labelled by gold particles (Fig. 4b), while clusters of interchromatin granules and the cytoplasm are unlabelled, except for the mitochondria. Again, the quantitative analysis have revealed that the average density of the hsiPG labelling, although lower than the condensed chromatin, is significant compared to that of cytoplasm (Table 3). After these two immunocytological methods, we used a cytochemical method, the osmiumammine Feulgen-like reaction, which offers the possibility to identify the DNA in the thickness of the section and which allows to study the organization of DNA present within the hsiPG (Derenzini, 1995). The cells, previously submitted to a heat shock during their culture and then reincubated at 37 °C, were stained with osmium-

202.71 ± 182.38* 688.33 ± 144.82** 3.58 ± 1.83

Mitochondria excepted.

ammine complex. To locate the possible deposition of osmium-ammine on hsiPGs, we used pairs of serial sections. The first section was mounted on a nickel grid and classically contrasted with uranyl acetate and lead citrate while the next section was mounted on a gold grid and stained with the osmium-ammine reaction. Two examples of serial section pairs (Fig. 5a,b and c,d) show the deposit of osmium-ammine on DNA containing structures, such as clumps of condensed chromatin. Regarding hsiPGs, they are not visible on the cell sections stained by the osmium-ammine reaction (Fig. 5a and c). However, no ‘‘empty holes” formed by the deposit of osmium-ammine on the surrounding condensed chromatin and which could indicate the location of a hsiPG, can be seen either (Fig. 5a or c). In few sections, some osmium-ammine-positive material can be detected at the place where hsiPGs are observed on classically contrasted sections (compare Fig. 5a,b and c,d). This material appears to be linked to the surrounding chromatin. It is also interesting to note that twisted DNA cordons are identified in hsiPG-containing areas stained with the osmium-ammine reaction (Fig. 5c, insert). 4. Discussion PGs are structures very difficult to investigate, because of their small size, their dispersion and their small number in mammalian cell nucleus. To facilitate their study, we submitted our HeLa cell cultures to a hyperthermal shock, a treatment known to increase the PG number in cells (Cervera and Montero, 1980; Mähl et al., 1989). Under these experimental conditions, we have shown that the hsiPG number per nuclear area considerably increases, leading essentially to cluster formation in certain nuclear regions, particularly in large blocks of condensed chromatin associated with nucleoli and nuclear envelope. These clusters have a remarkable organization, in a honeycomb form, in which each hsiPG seems connected to the chromatin walls by a stalk. In some cluster centres, a fibrillar structure, recalls the stress-induced nuclear bodies described previously (Chiodi et al., 2000), is also present. Under these optimal conditions for observing the hsiPGs, we analysed their content in nucleic acids using cytochemical and immunocytological methods applied on ultrathin sections. 4.1. hsiPGs contain RNA but also DNA The use of an immunocytological technique involving two antiRNA antibodies has allowed us to demonstrate the presence of RNA in hsiPGs. Previously, other methods for detecting RNA, such as autoradiography (Puvion et al., 1981), RNases-gold complexes (Cheniclet and Bendayan, 1990), in situ hybridization (Visa et al., 1993a,b) and bromouridine incorporation in cells (Chiodi et al., 2000), have suggested the presence of RNA in PGs. However, these methods present several drawbacks. Indeed, the autoradiography resolution (about 100 nm) is greater than the maximum size of a PG. With regard to the bromouridine incorporation technique, it

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Fig. 5. (a–d) Cytochemical detection of DNA within hsiPG clusters. Detection of DNA within hsiPG cluster from HeLa cells exposed to 42 °C for 60 min and then reincubated for 120 min at 37 °C as revealed with the Feulgen-like osmium-ammine staining (Fig. 5a and c). The chromatin clumps (C) are strongly stained. In nuclear areas containing hsiPGs as revealed with the corresponding serial section classically contrasted with uranyl acetate and lead citrate (Fig. 5b and d), we cannot see granules or empty cells in which the hsiPGs could reside after the osmium-ammine reaction. Arrows indicate hsiPGs as observed on a classically contrasted section and the corresponding site on the serial section stained with the osmium-ammine complex. These sites contain osmium-ammine-positive material connected with the surrounding condensed chromatin. Insert: higher magnification of the enclosed area in the Fig. 5c showing twisted DNA cordons. Bars = 0.1 lm.

used gold particles of 10 nm in diameter rather than 5 nm, which diminishes the labelling precision. Furthermore, this technique does not consider all the RNAs but only those synthesized during 30 min of contact with the precursor. On the other hand, we must note that most of these techniques were not subjected to a quantitative study that could significantly confirm the RNA content of PGs. Nevertheless, Cheniclet and Bendayan (1990) quantified the PGs labelled by one or more gold particles and estimated that over 20% of PGs contained RNA. This value is similar to that obtained here with our two anti-RNA antibodies (26.6%). Furthermore, we have shown that the hsiPG labelling by gold particles is significant compared to background noise such a quantitative study was never done before. Thus, in addition to have used a technique very specific for RNA, we have achieved a quantitative evaluation of the hsiPG labelling which enables us to reinforce the notion that PGs contain RNA. Regarding the DNA detection in hsiPGs, we used three different techniques. The first two were immunocytological approaches characterized by high specificity. Both techniques provide a significant labelling over the hsiPGs compared to background noise. That is the first time that DNA is clearly identified in hsiPGs. Before only

one author (Si, 1993) has suggested the presence of human papillomavirus DNA over PGs in human carcinoma cells of the cervix by in situ hybridization but without quantitative study. Other authors who resorted to less sensitive methods, such as enzymatic digestions (Vazquez-Nin and Bernhard, 1971; Smetana et al., 1979) or even cytochemical methods (Moyne, 1973) were unable to demonstrate the DNA presence in PGs. The difficulty to identify DNA in these structures might be explained by the fact that the DNA amount in PGs is low and therefore the detection techniques have to be very sensitive. So our labellings obtained with two sensitive and very specific methods and their quantitative analyses strongly suggest the presence of DNA in hsiPGs of HeLa cell nuclei. To attempt to obtain additional information about the DNA organization within hsiPGs, we used a cytochemical technique, the osmium-ammine staining. On sections stained with osmiumammine passing through hsiPG clusters, we have never observed hsiPGs or empty spaces that could harbor hsiPGs. Nevertheless, in the centre of some spaces corresponding to hsiPG location on classically contrasted sections, we identified an osmium-ammine-positive material connected to the surrounding condensed

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chromatin. These observations suggest hsiPGs might contain a small amount of DNA in continuity with the surrounding condensed chromatin. In summary, our cytochemical and immunocytological studies support the view that in HeLa cells submitted to a heat shock, hsiPGs could not only contain RNA but also a small amount of DNA. 4.2. Functional significance of PGs The PG function still remains obscure. On the basis of their similarity in structure and composition with Balbiani granules of Diptera, it is generally suggested that PGs are structures involved in the transport and/or storage of mRNAs (Monneron and Bernhard, 1969; Vazquez-Nin and Bernhard, 1971; Puvion and Moyne, 1981; Fakan et al., 1984; Vazquez-Nin et al., 1996). However the presence of DNA in hsiPGs as suggested here, disagrees with this assumption. Indeed, although Balbiani granules are initially in contact with the DNA, they are generally free in the nucleoplasm and totally devoid of DNA (Daneholt, 2001). Also, unlike Balbiani granules, no PGs have ever been seen in the passage of nuclear pores (Vazquez-Nin and Bernhard, 1971; Mehlin et al., 1991). Some authors also suggest that PGs could be aberrant RNAs in a degradation process (Cervera, 1979; Puvion et al., 1981). Knowing that pre-mRNA splicing starts during transcription (Beyer and Osheim, 1991), disruption of the transcription and/or of an early stage of the RNA maturation could lead to a RNA blockage on its DNA matrix and generate the appearance of PGs. This notion is consistent with the fact that PG number increases when mammalian cells are treated with drugs inhibiting RNA metabolism (Derenzini and Moyne, 1978; Puvion et al., 1981; Lafarga et al., 1993), resulting in an aberrant RNAs production. In this case, PGs should be located at transcription sites and/or in their immediate vicinity. However, incorporating bromouridine to study RNA dynamics in heat-shocked cells has revealed that hsiPG labelling required a long-time contact with the precursor (Chiodi et al., 2000). Thus, the hsiPGs cannot constitute transcription sites, since this phenomenon occurs within minutes, and this disagrees with the potential aberrant RNA nature of hsiPGs blocked on its DNA matrix. So, as neither of these two hypotheses satisfies all the experimental data, other experiments will be necessary to determine the role of PGs in the cell nucleus. Acknowledgments The authors acknowledge the skilful technical provided by F. Skivée. This work received financial support from the ‘‘Fonds de la Recherche Scientifique Médicale” (Grant No. 3.4540.06). FL and NT are PhD grant holders of the F.N.R.S. References Alverca, E., Franca, S., Díaz de la Espina, S.M., 2006. Topology of splicing and snRNP biogenesis in dinoflagellate nuclei. Biol. Cell 98, 709–720. Bendayan, M., 1984. Protein A-gold electron microscopic immunocytochemistry: methods, applications, and limitations. J. Electr. Microsc. Tech. 1, 243–270. Bernhard, W., 1968. A method of regressive coloration with use of the electron microscope. C. R. Acad. Sci. Hebd. Séances Acad. Sci. D 267, 2170–2173. Beyer, A.L., Osheim, Y.N., 1991. Visualization of RNA transcription and processing. Semin. Cell Biol. 2, 131–140. Biggiogera, M., Fakan, S., 1998. Fine structural specific visualization of RNA on ultrathin sections. J. Histochem. Cytochem. 46, 389–395. Biggiogera, M., Pellicciari, C., 2000. Heterogeneous ectopic RNP-derived structures (HERDS) are markers of transcriptional arrest. FASEB J. 14, 828–834. Cervera, J., 1979. Effects of thermic shock on HEp-2 cells. II. Inhibition of induction of perichromatin granules by cordycepin and actinomycin. D. J. Ultrastruct. Res. 66, 182–189. Cervera, J., Montero, M.R., 1980. Effects of thermic shock on HEp-2 cells. III. Accumulation of perichromatin granules. J. Ultrastruct. Res. 71, 1–13.

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