Zur Feinstruktur isolierter Kernmembranen aus tierischen Zellen

Summary

A method developed for isolating pieces of nuclear envelopes from plant tissue (Franke, 1966) can also be applied to animal cells. The procedure consists of a treatment of highly purified nuclei with hypotonical shock and subsequent gentle sonication, followed by a fractionation of the nuclear membranes in a combined differential and discontinuous density gradient centrifugation. This method was successful with such diverse kinds of animal cells as mouse liver tissue and logarithmically growing Tetrahymena pyriformis, micronucleus-less strain GL. Suspensions of the isolated envelope pieces were examined with electron microscopical techniques, especially in negatively stained preparations.

• 1. The pieces of nuclear envelopes isolated from mouse liver show pore complexes with well developed annuli and inner annulus diameters of 65 ± 7 nm. In the majority of the pieces the frequency of the pores per nuclear surface unit is from 35 to 55 pores per μ². The annulus is composed of globular subunits with diameters in the range of 7 to 18 nm and more diffuse material which also extends into the pore's lumen. Testing the number of these subunits and their center-to-center-spacing with Markham's rotation-technique, it was found that in general either eight or nine subunits are arranged in an eight- or ninefold radial symmetry. This result considered together with the findings previously reported for onion root tip nuclei and some observations of other authors strongly suggests that the regular arrangement of eight or nine globular subunits in a circular pattern within the annulus is of widespread occurrence among the karyobions.

• 2. In the fraction of envelope pieces from Tetrahymena macronuclei two types, differing in the appearance of their pore complexes, can be distinguished. In both types extraordinarily high pore frequencies of 95 to 135 pores per μ² were counted. Type B, however, shows very well developed annuli around the pores with inner annulus diameters of about 20 nm, while almost no annular material is present in type A, where only the delineated perimeter of the pore shows up as negatively stained. The pore diameters of type A range from about 55 to 60 nm. It is assumed that these types represent different structural states of the pore complex relating to changes in the pore's functional role as a system regulating the nucleocytoplasmic interactions.

Zusammenfassung

Die Methode zur Isolierung von Zellkernmembranen aus pflanzlichem Gewebe (Franke, 1966) ist auch auf tierische Zellen anwendbar: von Mäuseleber wie von logarithmisch wachsender Tetrahymena pyriformis konnten Fraktionen gut erhaltener Kernhüllenstücke erhalten werden. Die isolierten Membranen wurden elektronenmikroskopisch untersucht (Negativkontrast). Zellkerne aus Mäuseleber haben eine Porenhäufigkeit von 35–55 Poren pro μ², der innere Annulusdurchmesser der Porenkomplexe beträgt 65 ± 7 nm. Der Annulus besteht aus 8 oder 9 globulären Untereinheiten mit Durchmessern von 7–18 nm, die streng radiärsymmetrisch angeordnet sind, und diffuserem Material, das sich auch in das Lumen der Pore hinein erstrecken kann. Makronuclei von Tetrahymena weisen eine bemerkenswert hohe Porenhäufigkeit von 95–135 Poren pro μ² auf. Man findet hier zwei Kernmembran-Typen, die sich vor allem im Ausbildungszustand des Annulus unterscheiden: Typ A (innerer Annulusdurchmesser etwa 55–60 nm) besitzt kaum Annulusmaterial, Typ B (innerer Annulusdurchmesser etwa 20 nm) ist durch einen äußerst stark entwickelten Annulus mit distinkten Untereinheiten charakterisiert. Diese Strukturunterschiede werden in Zusammenhang mit der Vorstellung von den Kernporen als Regulationssysteme der Kern-Cytoplasma-Wechselbeziehung diskutiert.