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