Differential labelling of the vegetative primary nucleus of the marine green alga Acetabularia by lectins, anti-immunoglobulin-antibodies and antinuclear autoimmunosera.

Abstract

The highly conservative ultrastructural morphology of nuclei in both unicellular and multicellular organisms gives rise to a general expansion of models for subnuclear assembling structures and their molecular functions originally valid for a single type of cell nucleus. Structural conservatism can be studied by the comparative analysis of the individual components forming the complex subnuclear structural entities. A structural homology is evident, for ex ample, if immunoglobulins or lectins specific for a nuclear antigen of one species cross-react with individual components of nuclei from diverse sources.Using antinuclear autoimmuno-antibodies of human origin, as well as some lectins and anti-immunoglobulin-antibodies (AIAs), for comparative analysis of nuclei by immunofluore scence microscopy we have determined structural homologies between nuclear components that belong to extremely diverse eukaryonts, namely, vertebrates and the unicellular marine green alga Acetabularia. Materials and methodsThe experiments were performed with isolated nuclei of the unicellular and uninucleate marine green alga Acetabularia. The algae were grown in Schreiber medium according to Hammerling (1944).After dipping a separated rhizoid in one of the prefixation agents (see below), the primary nucleus was squeezed out of the rhizoid. The isolated nucleus was immediately transferred to a glass slide coated with gelatine, or to an uncoated one, and was flattened by a silicone coated cover slide. After freezing in LN2 the cover glass was removed and the nucleus was postfixed in one of the postfixation agents (see below), each containing 3.5% formaldehyde.The flattened nucleus remained fixed to the glass support during all subsequent incubation and washing procedures, in contrast to non-flattened ones. However, without prefixation the nuclear structure was destroyed by the pressure of the cover slide. Nuclei, prefixed for 2 min and flattened in various salt environments, i.e., in "high salt" (100mM PBS, pH 7.2), "inter mediate salt" (50mM PBS, pH 7.2), or "low salt" (10mM PBS, pH 7.2), showed minimal morphological differences when analyzed in light microscopical phase contrast. Thus, the prefixation agent was structure conserving within a wide range of experimental conditions. Prefixation: The isolated nuclei were prefixed for 2min in FA-PBS-A1 (1.5% formalde hyde in W-PBS-1 (see below)), FA-PBS-A2 (1.5% formaldehyde in 100mM Na-K-phosphate buffer, 0.9% NaCl, 5% sucrose; pH 7.2), or FA-PBS-A3 (1.5% formaldehyde, 5% sucrose in W-PBS-3 (see below)).Postfixation: The nuclei were postfixed for 2 min in FA-PBS-B1 (3.5% formaldehyde in W-PBS-1 (see below)), FA-PBS-B2 (3.5% formaldehyde in 100mM Na-K-phosphate buffer,