[Preparation and characterization of nuclear matrix/chromosome scaffold in situ].

A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol.

[Intracellular localization of XCAP-E protein in XL2 (Xenopus laevis) cells under normal conditions and during inhibition of pRNA transcription and processing]. / Issledovanie vnutrikletochnoi lokalizatsii belka XCAP-E v kletkakh linii XL2 (Xenopus laevis) v norme i pri ingibirovanii transkriptsii i protsessinga pRNK

The interaction of condensin subunit XCAP-E with various nucleolar subcompartments in XL2 cells was studied. In the interphase cells, XCAP-E was associated with a granular component of nucleoli (as shown by double staining with antibodies against B23) and with small nucleolus-like structures in the nucleoplasm. Inhibition of transcription by actinomycin D does not disrupt interaction of XCAP-E with the granular compartment of segregated nucleoli. Treatment with DRB 5,6-dichloro-1 beta-ribofuranozide-benzimidazole causes disintegration of nucleolar fibrillar complexes, but does not affect nucleolar localization of XCAP-E. The data suggest that nucleolar association of XCAP-E is independent on the functional state of the nucleolus, and imply a possible role of this protein in rRNA processing and pre-fibosome assembly.

[Intracellular localization of XCAP-E and pEg7 condensins in normal mitosis and after the treatment inducing artificial changes in structural organization of mitotic chromosomes]. / Vnutrikletochnaia lokalizatsiia kondensinov XCAP-E i pEg7 v norme i pri vozdeistviiakh, vyzyvaiushchikh iskusstvennoe izmenenie strukturnogo sostoiianiia mitoticheskikh khromosom.

Function of condensin subunits XCAP-E and pEg7 (XCAP-D2) in the formation and maintaining of special organization of mitotic chromosomes has been studied in Xenopus laevis cells (XL-2). The experimental conditions involved blocking chromosomes being in the condensed state in cells treated by cytostatics, or during their reversible artificial decondensation. The latter was induced by incubation of living cells in hypotonic medium. In extensively mollen chromosomes, XCAP-E and pEg7, remained associated with axial regions of chromosomes. In contrast, upon adaptation of cells to hypotonic conditions and recondensation of chromosomes to nearly initial state, both proteins dissociated from chromosomes into the cytoplasm. In K-mitotic cells, after a 3-6 h treatment with nocodazole or taxol, considerable dissociation of XCAP-E and pEg7 from chromosomes was observed without significant changes in overall level of chromosome compactization. Taken together the data suggested that condensins play no important role in maintaining mitotic chromosomes being in condensed state. Rather, it seems probable that mitotic function of condensins may be associated either with the formation of the higher order chromosome structure, and/or segregation of sister chromatids, the processes being tightly linked with chromosome compactization. This paper is in memory of Professor Katherine Le Guellec of Rennes-1 University, who left us in June 2001. Professor Le Guellec initiated this work in Rennes and offered all the possible help that this work be continued in Moscow University. Let the memory of Katherine, a great scientist and sympathetic friend, live for ever in ours hearts.