Localization of nuclear antigens during preparation of nuclear matrices in situ.
Nuclear matrix structure closely resembles the organization of nonchromatin components of nuclei in situ. However, reports on the extent to which nuclear components are reorganized during matrix isolation have produced conflicting results, and the reality of an in situ nuclear matrix is still in question. We have prepared nuclear matrices by processing cells still attached to the growth substrate through the extraction steps, thus avoiding mechanical disruption due to homogenization and centrifugation. Furthermore, the extensive residual cytoskeleton seems to keep the residual nuclei "stretched out" so that they retain many features of intact nuclei. Indirect immunofluorescence staining was used to compare the distribution of nuclear antigens in intact nuclei with their organization in nuclear matrices, as well as at each stage of nuclear matrix preparation. We have applied monoclonal antibodies P1, I1, PI1, and PI2, which had been generated against isolated matrices, as well as autoimmune sera detecting lamins, perichromin, and centromere antigens. Chromatin and RNA extraction was monitored with Hoechst 33258, ethidium bromide, and antihistone. The lamins, PI1, and, to a great extent, PI2 and centromere antigens were little affected by the extraction. The data suggest furthermore that PI1 is a fundamental nuclear matrix component and may serve in integrating peripheral and internal nuclear functions. P1 and perichromin were extensively redistributed after chromatin extraction, supporting a role for these antigens in spatial ordering of chromatin. I1 was progressively extracted at each stage of nuclear matrix preparation and was artifactually associated with matrices which had not been digested with RNase. This study demonstrates unequivocally that the organization of many nuclear matrix components in final preparations reflects their organization in situ. It does indicate, however, that some components retained in matrices are extensively redistributed during nuclear matrix preparation and that their role in nuclear organization must be evaluated in consequence.
Chaly
N
N
Little
J E
JE
Brown
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D034961:Antigens, Nuclear; D002467:Cell Nucleus / Q000648:ultrastructure*; D002478:Cells, Cultured; D002503:Centromere / Q000648:ultrastructure; D004247:DNA / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D003902:Detergents; D006657:Histones / Q000302:isolation & purification; D051379:Mice; D008858:Microscopy, Phase-Contrast; D009698:Nucleoproteins / Q000032:analysis*; D017830:Octoxynol; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D012313:RNA / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000941:Antigens; D034961:Antigens, Nuclear; D003902:Detergents; D006657:Histones; D009698:Nucleoproteins; D011092:Polyethylene Glycols; D012313:RNA; D017830:Octoxynol; D004247:DNA; D003850:Deoxyribonuclease I