1
Localization of condensin subunit XCAP-E in interphase nucleus, nucleoid and nuclear
matrix of XL2 cells.
Elmira Timirbulatova, Igor Kireev, Vladimir Ju. Polyakov, and Rustem Uzbekov*
Division of Electron Microscopy, A.N.Belozersky Institute of Physico-Chemical Biology,
Moscow State University, 119899, Moscow, Russia.
*Author for correspondence: telephone. 007-095-939-55-28; FAX 007-095-939-31-81 e-mail: [email protected]
Key words: XCAP-E; nucleolus; condensin; nuclear matrix; Xenopus.
Abbreviations: DAPI , 4’, 6 diamidino-2-phenylindole; DNP, deoxyribonucleoprotein; DRB,
5,6-dichloro-1b-d-ribofuranosylbenzimidazole; SMC, structural maintenance of chromosomes; XCAP-E, Xenopus chromosome associated protein E. 2
Abstract
The Xenopus XCAP-E protein is a component of condensin complex In the present work we investigate its localization in interphase XL2 cells and nucleoids. We shown, that XCAP-E is localizes in granular and in dense fibrillar component of nucleolus and also in small karyoplasmic structures (termed “SMC bodies”). Extraction by 2M NaCl does not influence
XCAP-E distribution in nucleolus and “SMC bodies”. DNAse I treatment of interphase cells permeabilized by Triton X-100 or nucleoids resulted in partial decrease of labeling intensity in the nucleus, whereas RNAse A treatment resulted in practically complete loss of labeling of nucleolus and “SMC bodies” labeling. In mitotic cells, however, 2M NaCl extraction results in an intense staining of the chromosome region although the labeling was visible along the whole length of sister chromatids, with a stronger staining in centromore region. The data are discussed in view of a hypothesis about participation of XCAP-E in processing of ribosomal
RNA. 3
Introduction
During the last decades considerable progress has been made in the studies of the structural organization of eukaryotic chromosomes, especially at the lower levels of chromosome compactization (nucleosomes, 30-nm chromatin fiber). However, despite many efforts, principles of higher order chromosome structure and underlying molecular mechanisms remain a matter of debate. Recently, considerable interest has been drawn to a specific class of chromosomal proteins - SMC proteins. It was shown that SMC proteins participate in multiple processes of chromosome dymanics, including mitotic chromosomes condensation, sister chromatid cohesion and segregation, dosage compensation and DNA recombinant reparations (Hirano et al., 1997; Losada et al., 1998; Lieb et al., 1998; Stursberg et al., 1999; Strunnikov et al., 1993; Strunnikov et al., 1995; Jessberger et al., 1996). SMC proteins fell into two major sub-families, of which SMC2/SMC4 are believed to be mainly involved in chromosome compactization. The multisubunit complex consisting of
SMC2/SMC4 heterodimer together with regulatory proteins was shown to be required for mitotic chromosomes condensation, hence the term condensin (Hirano, 1999).
Recently some data emerged suggesting that condensin subunits may have exerted some additional functions during interphase. For example, condensin subunits were found in the nucleolus of interphase HeLa (Cabello et al., 2001) and XL2 cells (Uzbekov et al., 2003;
Timirboulatova et al., 2003). Cabello and colleagues (2001) suggested interaction of SMC proteins with perinucleolar chromatin where they would be involved in reorganizations of ribosomal DNA. This hypothesis is supported by data of chromatin immunoprecipitation experiments performed in S. cerevisiae condensins with ribosomal DNA (Freeman et al.,
2000). There is also an alternative hypothesis, based on the studies of condensin dynamics upon inhibition of transcription and processing of ribosomal RNA, suggests that condensin subunits might be involved in pre-ribosome assembly (Uzbekov et al., 2003; Timirboulatova 4 et al., 2003).
In addition, a subpopulation of condensin proteins hCAP-C and hCAP-E in human cells and XCAP-E in Xenopus laevis cells was detected in small intranuclear structures of unknown origin (Schmiesing et al., 2000; Uzbekov et al., 2003; Timirboulatova et al., 2003).
Colocalization of these SMC-positive structures with phosphorylated form of histone Í3 allowed a speculation that nuclear condensins associated with interphase chromosomes participate in re-initiation of mitotic chromatin condensation (Schmiesing et al., 2000).
However, some of these structures also displayed co-localization with nucleolar proteins
(Timirboulatova et al., 2003), making them similar to Cajal bodies – specialized nuclear structures where assembly of transcription machinery is thought to take place (Gall, 2000).
In the present work, we studied ultrastructural localization of condensin subunit
XCAP-E in interphase XL2 cells using immunogold technique, and character of its interaction with different structural components of cell nucleus and nuclear matrix. 5
Results
Localization of XCAP-E in interphase cells
In interphase XL2 cell nuclei usually have from 1 to 4 nucleoli. In each nucleolus usually contains 1 to 3 fibrillar complexes, consisting of fibrillar centers and dense fibrillar component. Fibrillar complexes are localized in the central area or displaced to the periphery of the nucleolus. The remaining volume is occupied by granular component (Fig 1).
To study localization of XCAP-E, two principal ways of cells preparation were used:
1) permeabilization in the 0.5 % Triton X-100 in PBS with subsequent fixation in 3 % paraformaldehyde in the same buffer, or 2) fixation in 3 % paraformaldehyde in PBS and the subsequent permeabilization in 0.5 % Triton X-100. Both methods yielded identical results; therefore permeabilized cells were used for immunolocalization of XCAP-E after enzymatic digestion of nucleic acids.
In XL2 cells, XCAP-E colocalized in nucleolus with B23 – the marker protein of granular component. Both proteins were localized in the peripheral zone of the nucleolus, forming a continuous sphere or one to three hemispheres circumventing fibrillar complexes (Fig. 2 a-d).
Besides nucleolus, in most of the nuclei antibodies to XCAP-E decorated small globular domains in karyoplasm (Fig. 2 a-d).
Fig.1 Ultrastructure of nucleolus in XL2 cell. FC –fibrillar center, DFC – dense fibrillar component, GC – granular component. Bar, 0.5 µm. 6
The number of these domains varied from 2 to 5 per nucleus, with the size ranging from 0.5 to 1 mm. Since another extranucleolar B23-positive domain is known to be Cajal bodies (Gall, 2000 and references therein) we performed double labeling of XL2 cells with antibodies to XCAP-E and coilin – the marker protein of Cajal bodies. Surprisingly, no co- localization of these proteins was found (Fig. 2 e-h). Thus, globular domains containing proteins XCAP-E and B23 are not an equivalent of Cajal bodies in these cells, and possibly represent a special type of intranuclear structures. As XCAP-E belongs to the family of SMC proteins (Hirano et al., 1997), here we term these structures SMC-bodies, for short.
Fig. 2 Colocalization of XCAP-E with nucleolar protein B23 or coilin in interphase XL2 cells. Cells were grown and fixed as described under MATERIALS AND METHODS and processed for double immunofluorescence staining with anti-XCAP-E affinity-purified polyclonal antibodies and monoclonal antibodies against human B23 (a-d), or with anti-XCAP-E antibodies and monoclonal antibodies against coilin (e-h). In both cases cells were counterstained with DAPI for DNA visualization (a, d, e, h). Triple DAPI/XCAP-E/B23 labeling (d) and DAPI/XCAP-E/coilin labeling (h) are shown for colocalization of the proteins. Both XCAP-E and B23 were localized in nucleolar periphery as continuous sphere or one - three hemispheres circumventing fibrillar complexes. Besides nucleolus, antibodies to XCAP-E and B23 decorated small globular structures in karyoplasm (d, arrows). Coilin was localized in karyoplasm as numerous fine globular structures. However, in these structures colocalization of coilin with XCAP-E was not observed (h). Bar, 10 µm 7
Fig. 3 Immunoelectron microscopic localization of XCAP-E in control interphase XL2 cells and after actinomycin D and DRB treatments. Cells were grown as described in MATERIALS AND METHODS and fixed without any treatment (a) or after 4h incubation with 5 µg/ml actinomycin D (b) or after 4h incubation with 100 µg/ml DRB (c). After fixation cells were labelled with anti-XCAP-E affinity purified polyclonal antibodies and secondary anti-rabbit antibodies, conjugated with 1 nm gold particles, followed by silver enhancement. In control cells (a) high density of particles was observed within the granular component (GC) and dense fibrillar component (DFC) of the nucleolus. In fibrillar centers (FC) the labeling was absent. After actinomycin D treatment (b) nucleoli were segregated into granular and fibrillar compartments. Labeling was found in granular compartment, but not in fibrillar compartment of segregated nucleolus. In fibrillar compartment the labeling is absent. In cells after DRB treatment (c) XCAP-E labeling was localized in granular component (GC) and dense fibrillar component (DFC) of segregated nucleolus, whereas the fibrillar centers were devoid of gold label. Bar, 0.5 µm.
In a previous paper we reported ultrastructural localization of some condensin subunits using colloidal gold-labeled antibodies (Uzbekov et al., 2003). This approach proved to be rather ineffective due to restricted accessibility of antigens in such a dense structures as nucleolus. In the present work, improved procedure was used, which includes aldehyde fixation in combination with NanoGold-labeled antibodies and silver enhancement. This method is more effective for structure preservation while eliminating problems with antigen accessibility. 8
The immunoelectron microscopy analysis of XCAP-E distribution showed localization of this protein in granular and dense fibrillar component of nucleolus, but not in fibrillar centers. In some cases, dense fibrillar component not only circumvented fibrillar centers, but also penetrated into fibrillar centers as electron-dense fibers (see Fig. 3 a).
Besides nucleolus and “SMC bodies”, weak labeling of XCAP-E in karyoplasm was observed. At an ultrastructural level, immunogold labeling using antibodies against XCAP-E showed distribution of this protein throughout the nucleus in connection with DNP-fibrils
(Fig. 3 a).
Fig. 4. Immunofluorescence localization of XCAP-E in interphase cells after DNAse or RNAse treatment. Cells were grown and fixed as described under MATERIALS AND METHODS and processed for immunofluorescence staining with anti-XCAP-E affinity purified polyclonal antibody after DNAse (a-c) or RNase (d-f) treatments. In both cases cells were stained with DAPI for DNA labeling (a,c,d,f). Double DAPI/XCAP-E labeling is shown (c,f). Complete DNA hydrolysis was found throughout the volume of nucleolus (b,c). Exit of this protein in cytoplasm was observed. After treatment on permeabilized cells with RNAse XCAP-E was found in cytoplasm and did not found in nucleolus. Bar, 10 µm 9
Suppression of rRNA synthesis and processing, by actinomycin D and DRB respectively, causes segregation of nucleoli into structurally separated compartments.
However, XCAP-E remained associated with remnant nucleoli (Fig. 3 b, c). Interestingly, in nucleoli segregated under the effect of actinomycin D, XCAP-E was detected in association exclusively with granular component, while after DRB treatment, XCAP-E was found both in granular and dense fibrillar components (Fig. 3,c).
For identification of molecular components with which, presumably, XCAP-E can be associated, permeabilized cells were pretreated with nucleases (DNAse I or RNAse A) before immunostaining. DNAse I digestion resulted in complete hydrolysis of DNA, as monitored by staining with DNA-specific fluorochrome DAPI (Fig. 4 a-c). Under these conditions, staining with antibodies to XCAP-E resulted in strong homogenous labeling not only of peripheral regions, but also throughout the whole nucleolus. In addition, substantial increase of cytoplasmic staining intensity was detected. After RNAse treatment, XCAP-E was not found in association with remnant nucleolus, while cytoplasmic staining become even more prominent, as compared to DNAse-treated cells (Fig. 4 d-f).
Localization of XCAP-E in nucleoids and nuclear matrix.
Nucleoids prepared under the conditions stabilizing nuclear matrix had typical structure: they were represented by nuclear lamina, residual nucleoli and fibro-granular network, surrounded by halo of DNA fibers extruded from the nucleus (Cook and Brazell,
1975; Lebkowski and Laemmli, 1982; Gerdes et al., 1994; Sheval and Polyakov, 2002) (Fig.
5 a-d). Under these conditions, remnant nucleoli were surrounded by dense DAPI-positive material (Fig. 5 a). In nucleoids, XCAP-E and B23 colocalized in residual nucleoli and SMC bodies (Fig. 5 b-c). 10
Fig. 5 Immunofluorescence localization of XCAP-E and B23 in nucleoids after nucleases treatments. Nucleoids were prepared as described under MATERIALS AND METHODS and processed for double immunofluorescence staining with polyclonal anti-XCAP-E and monoclonal antibodies against B23 withouth additional treatments (a-d), or after DNAse (e-h) or RNAse (i-l) treatments. Cells were stained with DAPI for DNA labeling (a, e, i). Triple DAPI/XCAP-E/B23 labeling was shawn (d, h, l). Nucleoids had typical structure: they were represented by residual nucleus with fibrillar-granular network of DNA-containing material and halo of DNA extruded from nucleus (a). The XCAP-E labeling was visible on periphery of nucleolus as one to three hemispheres or rings; SMC bodies preserved the size and shape (arrows). After extraction with 2M NaCl XCAP-E partially leaved the nucleus, but did not associated with nuclear halo. XCAP-E and B23 were co-localized in residual nucleoli and SMC bodies of nucleoids. After treatment with DNAse (e-h), nucleoids were not stained with DAPI (e). Antibodies to XCAP-E and B23 labeled nucleoli similar to non-extracted nuclei, however intensity of fluorescence was weaker than in "intact" nucleoids. Significant quantity of both XCAP-E and B23 was observed in cytoplasm. After RNAse treatment zones of condensed chromatin, intensively labeled by DAPI were seen around nucleoli and SMC bodies (i). XCAP-E labeling was very weak in nucleolus and relatively strong in cytoplasm (j). B23 was not detected in nucleolus (k), but strong labeling in cytoplasm was observed. Image overlapping showed that XCAP- E did not colocalize with condensed chromatin. Bar, 10 µm. 11
Similar to control non-extracted cells, the labeling was detected on the periphery of nucleolus as one to three hemispheres or rings; SMC bodies retained the same size and shape, as in control. Image overlapping showed, that DAPI-positive material and XCAP-E did not colocalize neither in nucleolus, nor in SMC bodies (Fig. 5 d).
After treatment of nucleoids with DNAse I, nuclei became virtually DAPI-negative
(Fig. 5 e-h). Antibodies to XCAP-E and B23 stained nucleoli the same way as in control cells. However, intensity of fluorescence was weaker compared to "intact" nucleoids.
Significant fluorescence observed in cytoplasm can be explained by partial release of XCAP-
E and B23 from nucleus.
After treatment of nucleoids with RNAse zones intensely labeled with DAPI were formed around nucleoli and SMC bodies (Fig. 5 i-l). Labeling with antibodies to XCAP-E was significantly reduced compared to control preparations, while cytoplasmic fluorescence became relatively strong. B23 was practically undetectable in remnant nucleoli, but strong labeling in cytoplasm was observed. On merged images it is clearly seen, that XCAP-E is not co-localized with DAPI-positive zones around nucleoli (Fig. 5).
Localization of XCAP-E in chromosomal scaffolds
In control cells, since early prophase, antibodies to XCAP-E stained condensed chromosomes. In metaphase, XCAP-E appears in axial areas along the whole length of sister chromatids, showing punctate patterns (Fig. 6 a-c). The labeling was present also in spindle pole areas (Timirbulatova et al., 2002).
In mitotic cells, permeabilized in conditions stabilizing chromosomal scaffold and extracted by 2M NaCl, staining with DAPI revealed residual chromosomes as long thin fibers
(Fig. 6 d-f). 12
Fig. 6 Immunofluorescence localization of XCAP-E in chromosome scaffolds. Cells were treated and fixed as described under MATERIALS AND METHODS and processed for immunofluorescence staining with anti-XCAP-E affinity purified polyclonal antibodies. Normal prometaphase cells (a-c) permeabilized in conditions stabilizing scaffolds and extraxted by 2M NaCl (d-f) are shown. Cells were stained with DAPI for DNA visualization (a, d). In control cell (a-c) XCAP-E was localized in axial regions along entire chromatid as a series of discrete dots, with higher concentration in centromeric regions; in extracted cells (e, f) partial release of XCAP-E into halo was observed. In both cases antibodies to XCAP-E additionally decorated mitotic poles. Bar, 10 µm.
In prophase and anaphase, antibodies to XCAP-E labeled sister chromatids homogeneously, from middle metaphase till the beginning of segregation of chromatids to the poles, more intense fluorescence in centromeric areas (data not shown) was clearly seen.
After such a treatment spindle pole staining was also preserved (Fig. 6). 13
Discussion
XCAP-E – a component of multisubunit condensin complex, was isolated from
Xenopus laevis egg extract. Alongside with other condensin subunits, XCAP-E was localized by immunocytochemical methods in axial regions of mitotic chromosomes (Hirano, 1998;
Cubizolles et al., 1998, Maeshima and Laemmli, 2003).
Detection of condensins in nucleolus and SMC bodies shows that function of these proteins can’t be limited to mitotic chromosome compactization or other processes of chromosome dynamics. For understanding functional role of condensins it is of importance to follow the spatial dynamics of these proteins in interphase and mitosis.
In the present work precise immunolocalization of XCAP-E to the main sub-domains of interphase nucleus was performed. Immunogold labeling using antibodies to XCAP-E had shown that in nucleoli of cells fixed in situ this protein is located in granular component and in dense fibrillar component whereas fibrillar centres always remain free of labeling. Similar distribution of XCAP-E was observed in nucleolus after inhibition of rRNA synthesis and processing. Based on these data, it is possible to conclude that XCAP-E does not interact with the activated genes of ribosomal RNA located in fibrillar centers, as has been proposed earlier (Freeman et al., 2000; Cabello et al., 2001).
The question remains, with which nucleolar components (DNA, RNA or proteins)
XCAP-E interacts during interphase. It is known that formation of pre-ribosomal particles is a vector process directed from fibrillar complexes towards the periphery of nucleolus, and initiated with the transcription of rDNA. Data concerning exact localization of rRNA transcription sites are controversial: according to some data expression of ribosomal genes takes place on the border between dense fibrillar component and fibrillar center (Dundr and
Raska, 1993; Mosgoller et al., 1998), the others suggest that it occures in discrete compartments in dense fibrillar component (Stanek et al., 2001; Koberna et al., 2002) or in 14 fibrillar centers (Thiry et al., 2000; Mais, Scheer, 2001). Processing of newly transcribed nucleolar RNA begins in dense fibrillar component and is finished in granular component
(Shaw et al., 1995; Ñmarko et al., 2000). Fibrillarin, a marker protein of early stages of rRNA processing is localized in dense fibrillar component (Puvion-Dutilleul and Christensen,
1993). It was earlier shown that in nucleolus of XL2 cells XCAP-E was never colocalized with UBF and perfectly colocalized with B23 – a protein of granular component (Uzbekov et al., 2003). Partial overlapping of signals from XCAP-E and fibrillarin was also observed, which could be attributed to a limited spatial resolution. Here we confirm, on the basis of immunoelectron microscopic data, that XCAP-E is indeed localized in the zone of dense fibrillar component. Thus, XCAP-E seems to accompain rRNA through the processing and pre-ribosome assembly. The fact that inhibition of transcription under the effect of actinomycin D leads to a complete separation of XCAP-E from dense fibrillar component points to its association exclusively with released full-sized transcripts rather than with nascent ones.
In vitro experiments demonstrated the ability of SMC proteins to bind DNA.
Moreover, chromatin immunoprecipitation assay data suggested specific interaction of SMC proteins with rDNA (Freeman et al., 2001). Since presence of DNA in granular component of nucleoli was previously detected at the ultrastructural level using osmium amine staining
(Derenzini et al., 1987) and a terminal transferase labeling (Thiry et al., 2000) it is tempting to suggest that localization of XCAP-E in granular component is due to its association with
DNA. However, analysis of interactions of XCAP-E with nucleolar components based on enzymatic digestion of intact and high-salt extracted nuclei showed that this association does not depend on the integrity of DNA. On the other hand, virtually complete dissociation of
XCAP-E upon removal of RNA could be interpreted as an evidence of its direct or indirect interaction with rRNA. 15
Initially, SMC protein ScII, a human homolog of XCAP-E was identified as a major component of chromosomal scaffold (Lewis and Laemmli, 1982). The procedure of in situ nuclear matrix preparation used in the present study revealed XCAP-E in association with scaffolds for mitotic chromosomes, in accordance with the data of Maeshima and Laemmli
(2003). We also showed that XCAP-E is an integral component of nucleoids, more specifically remnant nucleoli. Based on structural similarities of SMC with other coiled-coil proteins, many of them, like intermediate filament proteins, playing a structural role, one can suggest similar function of XCAP-E in nuclear matrix. Since the structural preservation of nuclear matrix is known to depend on integrity of RNA (Berezney et al., 1995), redistribution of XCAP-E after RNA digestion can be explained in two ways: either XCAP-E represents a structural integral component of remnant nucleoli, which disappears with the destruction of nuclear matrix, or is a peripheral component interacting with nucleolar matrix indirectly, through binding to RNA. Since, under chosen experimental conditions, we never observed complete disintegration of nuclear matrix and remnant nucleoli, the latter hypothesis seems more reliable.
The question about exact functional role of XCAP-E at different stages of ribosomal biogenesis as well as mechanisms of its action remains open, partly because, till now, there is no unequivocal opinion on the mechanism of action of SMC proteins. It was shown that condensins, in a complex with topoisomerases, induce ÀÒP-dependent positive supercoiling of cyclic DNA in vitro (Kimura and Hirano, 1997). It is possible that XCAP-E and topoisomerase I are involved in regulation of conformational changes of rDNA during transcription, which occurs in fibrillar centers and dense fibrillar component. However, the data that XCAP-E is major protein of granular component, and is absent from fibrillar centres, with only partial co-localized with fibrillarin in dense fibrillar component, makes this assumption improbable. 16
Alternatively, XCAP-E might somehow participate in shaping rRNA during formation of preribosomal particles. The latter does not necessarily mean that XCAP-E directly interacts with ribosomal RNA during processing. Probably, this connection is mediated by low-molecular weight RNA (snoRNA), which is known to take part in ribosomal biogenesis (Dundr and Misteli, 2001). Assembly of transcriptional and processing mashineries is believed to take place in Cajal bodies where many nucleolar components, including fibrillarin, B23 and some snoRNA were found (Gall, 2000). Apart from nucleoli,
XCAP-E was found in compact nuclear domains also containing fibrillarin and B23
(Timirboulatova, 2003; this study). Based on abovementioned considerations, we suggested these domains to correspond to Cajal bodies. However, they proved to be coilin-negative.
Sensitivity of these domains to RNAse treatment and resistance to DNAse I also rule out the possibility that they represent a Xenopus equivalent of sites of histone H3 phosphorilation, described by Schmiesing et al., (2000). Thus, we described here a novel type of nuclear domains we termed SMC-bodies. Functional role of these domains remains unknown and requires further investigation.
Materials and methods
Cell culture
Xenopus XL2 cells were cultivated on coverslips in Petri dishes in Leibovitz 15 medium (ICN) supplemented with 10 % fetal calf serum (Vector) plus 100 µg/ml streptomycin and 100 µg/ml penicillin and 25 µg/ml amphotericin (Life Science
Biotechnology) at 25°C. 17
Nucleoids preparation
Nucleoids were prepared according to Sheval and Polyakov (2002). Cells were quickly washed with PBS, then permeabilized in 0.5 % Triton X-100 in the buffer A (50 mM TRIS-
HCl (pH 7.4), 5 mM MgCl2, 1 mM CuSO4, 1 mM PMSF) for 10 min. Then cells were washed in the same buffer without Triton X-100 and extracted in buffer B (2M NaCL, 10 mM EDTA,
20 mM TRIS-HCl (pH 7.4)) for 6 min. Cells were fixed in 3 % paraformaldehyde in buffer B and processed for immunocytochemical labeling.
Nuclease treatments
Cells, permeabilized in 0.5 % solution Triton X-100 in PBS or cells treated in conditions of stabilized nuclear matrix were used for enzymatic treatments.
Cells, permeabilized in 0.5 % Triton X-100 in PBS, were treated with DNAse I
(Boehringer Mannheim) in concentration of 250 µg/ml in PBS with 5 mM MgCl2, or RNAse
A (Sigma Biochemical) in concentration of 200 µg/ml in PBS, for 30 min at 37o Ñ. Cells were subsequently washed with PBS and fixed in 3 % paraformaldehyde in PBS for 10 min.
Cells, permeabilized in the buffer A, were treated with 250 µg/ml DNAse or 200
µg/ml RNAse in the same buffer for 30 min at 37o Ñ, when extracted with buffer B for 6 min and fixed 3 % paraformaldehyde in buffer B.
Immunocytochemistry
Cells were fixed in situ or after various treatments and incubated in PBS with 3 %
BSA 30 min at room temperature. Further cells were incubated with purified polyclonal antibodies against XCAP-E (rabbit) (1:50) (Uzbekov et al, 2003) and monoclonal antibodies against human B23 (1:100) (gift from Prof. T.I. Bulycheva, National Center for Hematology
RAMS, Moscow, Russia; Dergunova et al., 2000) or in purified polyclonal antibodies against
XCAP-E and antibodies against coilin (1:100) (kindly provided by Prof. M. Bellini) in PBS 18 containing 1 % BSA during 45 min at 37o Ñ. Then cells were washed 3 times 10 min in PBS and were incubated with the secondary anti-rabbit antibodies, conjugated with TRITC (Sigma
Biochemical) and anti-mouse antibodies, conjugated with FITC (Sigma Biochemical) 35 min at 37o Ñ, diluted 1:50 in PBS containing 1% BSA. Cells were washed with PBS 3 times 10 min and stained with 0.5 µg/ml DAPI (4’, 6 diamidino-2-phenylindole) (Sigma Biochemical) for 10 min, and mounted in Mowiol (Calbiochem). Samples were observed and photographed in microscope Opton III (Carl Zeiss), equipped with Neofluar 63/1.25 objective.
Immunoelectron microscopy
Control cells and cells treated with 5 µg/ml actinomycin D or 100 µg/ml DRB during
4 hours were fixed in situ with 3 % paraformaldehyde in PBS for 10 min. Cells were blocked in the 0.1 M phosphate buffer (pH 7.2) containing 3 % BSA for 30 min at room temperature.
Then cells were incubated in a solution of antibodies against XCAP-E (1:50) on the phosphate buffer containing 1 % BSA for 45 min at 37o Ñ, washed by PBS 3 x 10 min and incubated with the secondary goat anti-rabbit antibodies, conjugated with 10-nm colloidal gold particles (Sigma) diluted 1:50, or goat anti-rabbit Nanogold antibodies (Nanoprobes) diluted 1:200 in PBS containing 1 % BSA for 12 hours at 4o Ñ.
After labeling cells were washed in PBS 3 x 10 min. Then cells were fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), washed 3 x 5 min with distilled water, incubated in 1 mg/ml NaHB4 2 times 10 min at room temperature, and finally washed 3 x 5 min with distilled water. Silver enhancement of Nanogold labeling was perform according to
Danscher et al (1987).
Cells were dehydrated in ethanol of increasing concentrations and embedded in Epon
812. Ultrathin sections were cut on Reichert-Jung Ultracut ultratome (Reichert), stained with lead citrate and 2 % uranyl acetate and examined and photographed in HU-11B electron microscope (Hitachi). 19
Acknowledgements
We are grateful to Prof. M.Bellini (Univ. of Illinois at Urbana-Champaign, USA) and
Prof. F.Gianguzza (University of Palermo, Italy) for helpful discussion and critical reading of the manuscript. We also gratefully acknowledge the generous gifts of polyclonal anti-XCAP-
E antibodies from Dr. V. Legagneux, Prof. M.Bellini for anti-coilin antibodies, Dr. T.I.
Bulycheva (National Center for Hematology RAMS, Moscow, Russia ) for antibody against
B23, and Prof. J. Tata for XL2 cells (Mill Hill-NIMR Laboratory, London). We also give thanks to V.V. Kruglyakov for the invaluable help in electron microscopy.
This work was supported by RFBR grants 01-04-49287 to I.Kireev and 03-04-06396 to E.Timirbulatova and 03-04-48955-à to V.Yu. Polyakov
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