Localization of nuclear antigens during preparation of nuclear matrices in situ

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Abstract
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. Nuclear matrices were prepared 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. 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 were applied. 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 PI1 is apparently 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. The organization of many nuclear matrix components in final preparations reflects their organization in situ; some components retained in matrices are extensively redistributed during nuclear matrix preparation and that their role in nuclear organization must be evaluated in consequence.