Demonstration of a DNase-sensitive network associated with the nuclear pore complexes in rat liver nuclei

Abstract

Rat liver nuclei have been studied by transmission electron microscopy after resuspension in a phosphate-buffered salt solution containing SO2-4 as the quantitatively dominant anion. Owing to the high solubility of chromatin in the presence of SO2-4 instead of Cl- at isotonicity, nuclei are depleted for chromatin by DNase I digestion in this buffer, eliminating the need for high-salt extraction. This shows that at least 75% of the nuclear pore complexes are associated with fibrogranular structures, which ramify as a network throughout the nucleus, interconnecting the nuclear lamina, interchromatin granule clusters and nucleoli. Perichromatin granules are located in this material proximal to the nuclear pore complexes. Most of the chromatin is removed without major impact on the network, but below a level of 25% residual chromatin there is a considerable reduction of this material, and only about 15% of the connections to the nuclear pore complexes are resistant to digestion with DNase I or streptodornase A and B. The percentage of nuclear pore complexes connected to the network is further reduced by salt extraction and RNase treatment. These results suggest that DNA is an integral part of the network, which presumably plays a role in nucleo-cytoplasmic transport of RNA and protein.

Experimental observations of a nuclear matrix

“…The resulting preparations share similar morphologies, which are preserved to varying degrees. Methods that avoid detergents and/or are performed without hypertonic salt concentrations have been developed (Engelhardt, 1999;Jackson and Cook, 1988;Mirkovitch et al, 1984;Nickerson et al, 1997;Wan et al, 1999) and reveal ultrastructural features similar to those seen in traditional matrix preparations. The ultrastructure of the isolated nuclear matrix cannot, therefore, be ascribed, as critics assert, to NaCl-or Triton-induced precipitations of nuclear, but not cytoplasmic, proteins (Pederson, 2000).…”

Patrick W. Tank (1950–2012)

“…“The mode of association chromatin with nuclear envelope may involve the attachment of initiation sites of the replicating DNA to the pore complexes” 3. Similar interactions between chromatin DNA and nuclear pores have been reported recently 5, 6.…”

“…Many modern researchers come to conclusion that nuclear envelope is not only barrier, separating nucleoplasm from cytoplasm, and nuclear pores are not only channels linking these area. The idea about localization of the initiation transcription and replication sites on nuclear envelope intimately in contact with nuclear pores finds the support in modern papers 5, 24.…”

The Nuclear Matrix: Fractionation Techniques and Analysis

“…The resolution attained with the electron microscope has allowed detailed visualization of the fibrillar protein network within the nucleus of higher eukaryotic cells (Capco et al 1982;Fey et al 1986;Jackson and Cook 1988). The nuclear matrix has been viewed after RNase digestion (Berezney and Coffey 1974), DNase digestion (Capco et al 1982), and removal of chromatin by electroelution (Jackson and Cook 1988) and in a range of buffer conditions designed to minimize artifacts (Mirkovitch et al 1984;Jackson and Cook 1988;Nickerson et al 1997;Engelhardt 1999;Wan et al 1999). The nuclear matrix has also been viewed in paraformaldehyde-fixed sections of unextracted nuclei, identifying protein-rich interchromosomal areas consistent with the description of a nuclear matrix (Hendzel et al 1999).…”