ISOLATION OF A NUCLEUS‐ASSOCIATED MICROTUBULE SCAFFOLD FROM SPIROGYRA (ZYGNEMATALES, CHAROPHYCEAE)

A phragmosome‐like perinuclear scaffold attaches to helical chloroplast bands in the peripheral cytoplasm of Spirogyra crassa Kützing by rigid, stalk‐like cytoplasmic strands. Balanced integration of forces within this scaffold by microfilament‐based tension conveyed along stabilized microtubules (MTs) within the stalks controls transverse centering of the lenticular nucleus in the cylindrical cell prior to mitosis. Nuclei with their associated scaffolds were released from rapidly frozen, shattered cells upon thawing in a MT‐stabilizing buffer and could be purified by a procedure combining filtration, differential centrifugation, and isopyknic centrifugation in a linear density gradient. In the osmotically indifferent gradient medium Nycodenz™, nuclei with adhering scaffolds attained a buoyant density of 1.26–1.27 g·cm −3 . The nuclei maintained their energetically unfavorable, lenticular shape in vitro and appeared structurally preserved as judged by video‐enhanced differential interference contrast and by fluorescent staining of DNA with 4′‐6′‐diamidino‐2‐phenylindole. The stalks of the scaffold, though still radiating from the nuclear rim, lost their straightness and were often severed during the isolation process. The distal ends of many unsevered stalks and of stalk fragments were still associated with chloroplast fragments. Persistent flexural rigidity of the scaffold stalks and indirect immunofluorescence indicated preservation of MTs. In addition to tubulin, both actin and an intermediate filament antigen of M r ≈ 68 kDa were detected in the isolated nuclei with associated scaffolds by immunoblot with monoclonal antibodies.