[Nuclear matrix-lamina-intermediate filament system in PtK 2 cells].

Selective extraction, whole mount cell preparation and DGD embeddment-free section were involved in visualizing the nuclear matrix-lamina-intermediate filament system in PtK 2 cells. After extraction the anaphase chromosome residues adjoined to the cytoplasmic intermediate filament in some areas. Immunofluorescent staining showed us that the intermediate filament reacted with AE 1 and AE 3; McAb 223 could be localized specifically on the lamina while McAb C 23 could crossreact with cytoplasmic intermediate filament beside the lamina location, monoclonal antibody against lamin A (C) could also bind to chromosome residues. Antibody to 280 kD nuclear matrix protein which were positive stained in HeLa cells could not react with the nuclear matrix components of PtK 2 cells. 2-D electrophoresis demonstrated that there were some differences in the composition of the nuclear matrix-lamina-intermediate filament system of HeLa and PtK 2 cells. TdR treatment could lead to alteration of nuclear matrix proteins.

[The intermediate filament-lamina-nuclear matrix system of ES-M13 cells].

Sequential extraction and DGD embedment-free section TEM techniques were used as new methods to study the intermediate filament-lamina-nuclear matrix (IF-L-NM) system of cells. Murine embryonic stem cells (ES-M13) were investigated by means of electron microscopy, immunofluorescence microscopy and Western-blot analysis. There existed a delicate nuclear matrix network in the nucleus domain of detergent-extracted ES cells. The filaments of the nuclear matrix were found in close association with the nuclear lamina. Some intermediate filaments were also observed in the cytoplasm. In immunofluorescence microscopy, a bright, slightly granular cytoplasmic fluorescence, showing no polar concentration, was revealed with keratin monoclonal antibody AF5. We could not detect any significant fibrillar staining in the ES cells by indirect immunofluorescence method. With antibodies to vimentin and desmin, the ES cells showed only a nonspecific, weak fluorescence, similar to that seen in the control. In Western-blot analysis of electrophoretically separated polypeptides, three polypeptides with molecular weight of 65 KD, 62 KD and 52 KD, reactive with keratin monoclonal antibody were detected in the ES cells.

An immuno-electron microscopic study on the relationship between nuclear matrix and DNA in rat spermatocytes.

The nucleus of the mammalian spermatid undergoes a series of changes in its chromatin and nucleoprotein composition during transport from testis to epididymis. The sperm DNA is very tightly packaged by protamines instead of histones in somatic cells. However, the nuclear matrix and its association with DNA have not yet been definitively scrutinized with the electron microscope. The present study reveals that the protamine-depleted sperm nuclear matrix appears as a network of thick and thin filaments with glodular structures attached the these fibers. Monoclonal antibody to single- and doublestranded DNA was used to localize remnant DNA after extraction. By immunofluorescence microscopy, monoclonal antibody against DNA was localized outside the nucleus as a halo. Immuno-electron microscopy showed that gold particles were mainly associated with nuclear matrix surrounding the sperm head. Our results suggest a specific structural organization of sperm DNA with its matrix.

Fine structural observation of a nucleolar-nuclear matrix-lamina-intermediate filament system in transformed cells.

The nuclear matrix is a nonchromatin structure of the nucleus normally concealed by a much larger mass of chromatin. Several methods have been developed to remove chromatin from nucleus while preserving the underlying matrix architecture to some degree. The present study showed that after extraction of PtK2 cells and cervical carcinoma cells (CC3) with Triton X-100, ammonium sulfate and DNase, the nucleolar-nuclear matrix-intermediate filament (Nu-Nm-L-IF) network remained. The nucleolus was oval in shape and appeared as a fibrillar mass with an accentric dense fibrillar centre. This nucleolar skeleton was in direct connection with the nuclear matrix which in turn is connected with cytoplasmic intermediate filaments by lamins. It is concluded from these observations that the Nu-NM-L-IF system forms a continuous system which plays an important role in the maintenance of the nucleolar, nuclear and cytoplasmic integrity and cellular function.

Ultrastructural study on the development of the rat [correction of mouse] spermatocyte nuclear matrix during epididymal maturation.

The rat sperm nucleus, after sequential extraction with detergents, nuclease and ammonium sulfate, consists of a skeletal structure that resembles the original nuclear shape. This chromatin-depleted skeleton is formed by thick and thin fibers as well as globular structures of different sizes. These fibers form anastomosis. The sperm nuclei obtained from testis and caput epididymis exhibits a loose fibrous network with thin fibers at the center. The entire nucleus of the sperm in the caudal epididymis is formed by a dense network of thick and thin fibers. These highly branched matrix fibers had diameters of 35 and 12 nm. It is concluded that the increase in density of the matrix fibers is related to the condensation of the chromatin in the maturation of the spermatozoa.

Microwave fixation of nuclear matrix in tumor cells.

Microwave irradiation provides good fixation of human and animal tissues for light and electron microscopy. In this study, microwave irradiation was used for the fixation of cytoplasmic and nuclear matrix in tumor cells. The nuclear matrix appears well preserved and exhibits a network formed by thick and thin filaments. Hence microwave fixation can be used as a quick and effective method for the study of the morphology of nuclear matrix.

Immunogold labelling of the intermediate filament-lamina-nuclear matrix system in HeLa and BHK-21 cells.

Whole-mount, sequentially extracted cells combined with immunogold electron microscopy were developed to demonstrate the intermediate filaments, lamina, and nuclear matrix (IF-L-NM) and to identify their protein components. The IFs of HeLa cells were reacted both with keratin and vimentin monoclonal antibodies; meanwhile, the IF network of BHK-21 cell was reacted only with vimentin monoclonal antibody. The lamina and nuclear matrices of both HeLa and BHK-21 cell were labelled, respectively, with lamin monoclonal antibody-gold complex and 280 Kd nuclear matrix protein monoclonal antibody-gold complex. The monoclonal antibody to keratin could cross-react with the lamina both of HeLa and BHK-21 cells.