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N Stuurman

Publications and source records attributed to N Stuurman.

31 records · Page 2Linked to original sources
PubMed1995

Disassembly of the Drosophila nuclear lamina in a homologous cell-free system.

Stage 14 Drosophila oocytes are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins. Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. A previously unidentified soluble lamin isoform is easily seen after in vitro disassembly. This isoform is detectable but present only in very small quantities in vivo and is apparently derived specifically from one of the two interphase lamin isoforms. Cell-free nuclear lamina disassembly is ATP-dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. This system will allow enzymological characterization of cell-free lamina disassembly as well as molecular analysis of specific Drosophila mutants.

Adenosine Triphosphate↗
PubMed1995

Interphase phosphorylation of the Drosophila nuclear lamin: site-mapping using a monoclonal antibody.

The Drosophila nuclear lamin is highly phosphorylated during interphase. Two interphase isoforms, differing in degree of phosphorylation, can be distinguished by one-dimensional SDS-polyacrylamide gel electrophoresis. One migrates with an apparent mass of 74 kDa (lamin Dm1); the other is more highly phosphorylated and migrates as a 76 kDa protein (lamin Dm2). We generated a monoclonal antibody, ADL84 which binds to lamin Dm1 but not lamin Dm2. Binding of ADL84 to lamin Dm2 was restored by phosphatase treatment of immunoblots containing lamins. Immunoprecipitation with ADL84 demonstrated that purified Drosophila nuclear lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. Indirect immunofluorescence experiments suggest that lamin Dm1 is present in all Drosophila cell types. The epitope for ADL84 was mapped by analyzing binding to bacterially expressed lamin deletion mutants and subsequently by screening for point mutants (randomly generated by polymerase chain reaction) which were not recognized by ADL84. The ADL84-epitope encompasses amino acids R22PPSAGP (arginine 22-proline 28). Peptide competition experiments demonstrated directly that phosphorylation of serine 25 impedes lamin binding by ADL84. This suggests that serine 25 is the lamin Dm2-specific phosphorylation site.

Animals↗
PubMed1994

The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.

Animals↗
PubMed1993

Intranuclear filaments containing a nuclear pore complex protein.

Nuclear pore complexes (NPCs) are anchoring sites of intranuclear filaments of 3-6 nm diameter that are coaxially arranged on the perimeter of a cylinder and project into the nuclear interior for lengths varying in different kinds of cells. Using a specific monoclonal antibody we have found that a polypeptide of approximately 190 kD on SDS-PAGE, which appears to be identical to the recently described NPC protein "nup 153," is a general constituent of these intranuclear NPC-attached filaments in different types of cells from diverse species, including amphibian oocytes where these filaments are abundant and can be relatively long. We have further observed that during mitosis this filament protein transiently disassembles, resulting in a distinct soluble molecular entity of approximately 12.5 S, and then disperses over most of the cytoplasm. Similarly, the amphibian oocyte protein appears in a soluble form of approximately 16 S during meiotic metaphase and can be immunoprecipitated from egg cytoplasmic supernatants. We conclude that this NPC protein can assemble into a filamentous form at considerable distance from the nuclear envelope and discuss possible functions of these NPC-attached filaments, from a role as guidance structure involved in nucleocytoplasmic transport to a form of excess storage of NPC proteins in oocytes.

Animals↗
PubMed1992

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.

1-Methyl-3-isobutylxanthine↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Cell Membrane Permeability↗
PubMed1992

A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies.

We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus. By immunoblotting mAb 5E10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5. Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium. In the human bladder carcinoma cell line T24 the number of nuclear spots were found to be 21 +/- 10 (n = 132). In many cells spots were found also in the cytoplasm. In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots. Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes. The spherical structures that contain the antigen were tightly bound to the nuclear matrix. Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 microns diameter spherical, electron-dense structures, reminiscent of nuclear bodies. Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres. The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies. On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies.

Animals↗
PubMed1990

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins.

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.

Animals↗
PubMed1989

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.

Animals↗
PubMedDate not supplied

Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding.

Polymerization of intermediate filament proteins results from interactions among several distinct binding sites on the constituent proteins. Nuclear lamin head-to-tail polymers arise from one such interaction. We studied this binding using Drosophila lamin Dm0-derived fragments containing either the NH2-terminal or COOH-terminal binding site with a combination of co-immunoprecipitation, yeast two-hybrid, analytical ultracentrifugation, and electron microscopic assays. Fragment binding and full-length lamin head-to-tail polymerization were similar to each other in morphology, buffer requirements, and inhibition after phosphorylation with cdc2 kinase. Deletion analysis localized the binding sites to the ends of the rod domain that are highly conserved among all intermediate filament proteins. Point mutants, defective in binding, were isolated. Two were identical to point mutations in specific human keratin genes known to affect keratin assembly and to cause genetic skin diseases. Results further indicate that the binding sites only function in specific sequence contexts and that binding can be modulated by elements outside the binding sites (like the cdc2 kinase phosphorylation site). Our data indicate that one type of interaction in intermediate filament protein polymerization is the longitudinal binding of dimers via the conserved end segments of the coiled-coil rod domain.

Amino Acid Sequence↗