The nuclear scaffold or matrix is a mainly proteinaceous structure thought to act as a nucleoskeleton determining the higher order organization of eukaryotic chromatin. These structures are prepared from isolated nuclei by a series of extraction steps involving the use of ionic detergents or high salt, and restriction enzymes or non-specific nucleases to remove chromatin and other loosely bound components. Since these treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond to a nucleoskeleton existing in vivo. Recently, it has been demonstrated that the majority of nuclear matrix proteins are involved in RNA metabolism. In this study we have employed a morphological approach involving the use of confocal laser scanning microscopy and indirect immunofluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial distribution of two polypeptides involved in RNA metabolism, i.e., a 105-kDa component of spliceosomes and a ribonucleoprotein antigen. We demonstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compared with intact cells. Only when isolated nuclei were stabilized in vitro with the cross-linking agent sodium tetrathionate (NaTT) prior to extraction with 2 M NaCl and DNase I digestion, were the immunofluorescent patterns displayed by the nuclear matrix indistinguishable from those detected in intact cells. These results emphasize the usefulness of NaTT in studying putative nucleoskeletal structures, but also show that the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.
Changes in the subnuclear distribution of two RNA metabolism-related proteins can be detected in nuclear scaffold or matrix prepared by different techniques
NERI, Luca Maria;
1997
Abstract
The nuclear scaffold or matrix is a mainly proteinaceous structure thought to act as a nucleoskeleton determining the higher order organization of eukaryotic chromatin. These structures are prepared from isolated nuclei by a series of extraction steps involving the use of ionic detergents or high salt, and restriction enzymes or non-specific nucleases to remove chromatin and other loosely bound components. Since these treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond to a nucleoskeleton existing in vivo. Recently, it has been demonstrated that the majority of nuclear matrix proteins are involved in RNA metabolism. In this study we have employed a morphological approach involving the use of confocal laser scanning microscopy and indirect immunofluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial distribution of two polypeptides involved in RNA metabolism, i.e., a 105-kDa component of spliceosomes and a ribonucleoprotein antigen. We demonstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compared with intact cells. Only when isolated nuclei were stabilized in vitro with the cross-linking agent sodium tetrathionate (NaTT) prior to extraction with 2 M NaCl and DNase I digestion, were the immunofluorescent patterns displayed by the nuclear matrix indistinguishable from those detected in intact cells. These results emphasize the usefulness of NaTT in studying putative nucleoskeletal structures, but also show that the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
