Isolation and Characterization of Bovine Lymphocyte Nuclear Matrix

Abstract

ABSTRACT. A nuclear matrix was obtained from isolated nuclei of bovine lymphocyte by digestion with DNase I and subsequent extractions with 0.4 M and 2.0 M NaCl. 91.1 % of the nuclear protein, 99.5 % of the DNA and 83.1 % of the RNA were removed from isolated nuclei by these treatments. with the following nuclease digestion, almost all residual DNA and RNA was removed leaving the nuclear protein matrix.Ultrastructural analysis of the nuclear matrix revealed a spherical structure containing a peripheral lamina, an internal fibrogranular network and residual nucleoli.Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nuclear matrix demonstrated three major polypeptides of 68,000, 53,000 and 43,000 dalton as well as more than 50 minor polypeptides of various molecular weight classes. The 68,000 dalton polypeptide corresponds to lamin B, one of three predominant polypeptide components of rat liver nuclear matrix. However, polypeptides corresponding to lamin A and C were not detectable in the bovine lymphocyte nuclear matrix.Berezney and Coffey (4, 6) were the first to propose that a proteinaceous network, termed the nuclear matrix, serves as the skeletal foundation which determines the overall organization of rat liver nuclei. Since then, some proteinaceous structures, resembling a nuclear matrix, have been prepared from HeLa S3 cell (15), Tetrahymena (30), Friend erythroleukemia cell (19), Physarum polycephalum (25), 3T6 fibroblast (9) and others. These proteinaceous structures are very similar to each other, composed of three components : peripheral lamina, internal fibrogranular structure and residual nucleoli. The nuclear matrix appears to be a ubiquitous structure that is present in almost all eukaryotic cells. Much evidence is accumulating to suggest that the nuclear matrix not only may function as the skeleton of the nucleus but also may be associated with many dynamic aspects of nuclear function: nuclear swelling (30, 31), DNA replication (5,7,13,16,27), RNA transcription (1,11,23), hormone binding (3), T-antigen binding (9), processing and transport of RNA (22,29), etc.To know the real function of the nuclear matrix, it is important to examine the matrix in cells from a variety of organisms and tissues. In this paper, we first report the isolation method and characterization of the bovine lymphocyte nuclear matrix.Abbreviations: SDS, sodium dodecyl sulfate, PMSF, phenyl methyl sulfonyl fluoride; DEP, diethyl pyrocarbonate; GTM buffer, 50 mM Tris-HCl (pH 7.5) containing 25 % (v/v) glycerol and 2 mM MgCl2. 181

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

“…Lamin A/C and Lamin B1 form separate but interconnecting networks (Shimi et al 2008) and perform distinct functions at the nuclear lamina; nuclei deficient for Lamin A/C have decreased nuclear stiffness and increased deformations whereas nuclei deficient for Lamin B1 display normal nuclear mechanics (Lammerding et al 2006). Protein and lipid depleted nuclear matrices have been shown to reversibly shrink in response to bivalent cation concentration (Wunderlich and Herlan 1977) or nuclease treatment (Berezney and Coffey 1977;Nakayasu and Ueda 1981) and rat nuclei shrink in response to elevated calcium levels (Okada et al 2010). Degradation of nuclear material occurs after Lamin A/C depletion of the nucleus, as DNase deficient murine epidermis retains DAPI but not Lamin A/C staining in corneocytes (Fischer et al 2017).…”