N. Stuurman

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

Stabilization of the nuclear matrix by disulfide bridges: Identification of matrix polypeptides that form disulfides

The molecular structure of the nuclear matrix is still poorly understood. We have tried to assess which proteins are important structural elements by examining the process of stabilization of the nuclear matrix by sodium tetrathionate. Sodium tetrathionate stabilizes the nuclear matrix by oxidizing sulfhydryl groups to disulfides. We show that tetrathionate-stabilized matrices are disassembled in buffers containing SDS, indicating that the stabilized nuclear matrix is not a continuous network of cross-linked proteins. Using monobromobimane, a thiol-specific fluorescent reagent, we show that many protein thiols in the stabilized matrix are oxidized. By chromatography on activated thiol-Sepharose we estimated that about 50% of the matrix proteins had oxidized sulfhydryl groups. The protein composition of the material bound to activated thiol-Sepharose was similar to that of the not-bound material. A few proteins are highly enriched in the fraction that was bound to the column. This indicates that many matrix protein species are partially oxidized and that some proteins are completely oxidized. The oxidized protein thiols are found in relatively large complexes as determined by SDS gel-electrophoresis under nonreducing conditions. These results are interpreted in terms of protein-protein interactions in the matrix. The possible role of thiols and disulfides in the in vivo organization of the nucleus is discussed.

Induction of nuclear lamins A/C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells

Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired after induction of differentiation in vitro by certain drugs. In this study we have examined the time course of lamin A/C expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-1-methylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway.

Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells

A preembedment labeling procedure is described for the three-dimensional (3D) labeling of nuclear matrix proteins in permeabilized cells. The procedure is based on the use of ultra-small (1 nm) gold particles as a marker system. This marker penetrates the nucleus more efficiently than the conventionally used 5-10 nm colloidal gold probes. Dehydration is performed by freeze-substitution to preserve the ultrastructure of the cell as optimally as possible. During freeze-substitution the samples are stained by uranyl ions to stain the cellular material throughout the resin section. The 3D gold-labeled and uranyl-stained specimen is embedded in Epon resin and semi-thin (0.2-0.5 microns) sections are made for stereo electron microscopy. The applicability of this method is illustrated by the localization of nuclear matrix-associated nuclear bodies in permeabilized interphase and mitotic HeLa cells.