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On the fine structure of the frog's rod outer segments, observed by the freeze-etching technique

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Summary

The fine structure of the frog's (Rana esculenta) rod outer segments was investigated by two different methods: most of the experiments were made by means of the freeze-etching technique. The replicas were then examined by electron microscopy (40,000 X).

By means of a second method, rod outer segments were negatively stained prior to electron microscopy.

Inspection of the electron micrographs revealed that the frog's rod outer segments seem to be built up of three groups of “elongated structures” interpreted as fibrils (Fäden) arranged regularly at approximately equal distances. The diameters of the fibrils are below 100 Å; they depend on the state of light adaptation and on the chemical preparation before freeze-etching. The fibrils partly cross each other. In addition, there were found four groups of approximately equal distances between the fibrils. The order of magnitude of these spacings is from about 50 Å to a few hundred Å.

Negatively stained outer segments also reveal fibrils. The results are expressed in a working hypothesis consisting of two parts. It is supposed first that the core of the rod outer segment represents a three dimensional paracrystalline lattice (Raumgitter) of three different types of fibrils (d 1, d2, d4). The distances between the fibrils are interpreted as the lattice constants (a 1, a2, a3, a4). A unit cell of the lattice would consist of a web (Geflecht) of two different types of fibrils (d 1, d2) and four layers of parallel fibrils of the third type (d 4).

It is supposed, secondly, on the basis of a volume-evaluation, that the d1-fibrils contain rhodopsin, those of type d 2 another protein (not rhodopsin), and fibrils of type d 4 lipids.

The working hypothesis is supported by experimental findings of other authors (obtained by negative staining and diffraction of light and X-rays).

Attempts have been made to relate some electron micrographs of ultrathin sections to those of replicas. (Rosenkranz et al., 1969; Rosenkranz, 1969a.)

Zusammenfassung

Der Feinbau der Stäbchenaußenglieder des Frosches (Rana esculenta) wurde mit zwei verschiedenen Methoden untersucht: der größte Teil der Untersuchungen wurde mit der Gefrierätzmethode durchgeführt. Die Abdrucke (Masken der Bruchflächen) wurden im Elektronenmikroskop bei 40000facher Vergrößerung betrachtet.

Als zweite, von der ersten unabhängigen Methode, wurden Teile negativ kontrastierter Außenglieder des Frosches im Elektronenmikroskop betrachtet.

Die Auswertung der elektronenmikroskopischen Aufnahmen von Abdrucken ergab: die Außenglieder des Frosches scheinen aus 3 Gruppen „länglicher Gebilde“ aufgebaut zu sein, die in jeweils angenähert gleichen Abständen angeordnet sind. Die „länglichen Gebilde“ werden als Fäden bezeichnet; ihre Durchmesser liegen unter 100 Å. Die Größe der Durchmesser hängt vom Adaptationszustand und der chemischen Behandlung vor der Gefrierätzung ab. Die Fäden überkreuzen sich z.T. — Es wurden ferner 4 Gruppen angenähert gleicher Abstände zwischen den Fäden gefunden. Die Größe dieser Abstände liegt zwischen etwa 50 Å und einigen hundert Å.

Negativ kontrastierte Außenglieder ließen ebenfalls Fäden erkennen.

Die Ergebnisse werden zu einer zweiteiligen Arbeitshypothese zusammengefaßt. Im 1. Teil der Arbeitshypothese wird angenommen: der Innenkörper des Außengliedes (das ist das Außenglied ohne die erkennbare Zellmembran) ist ein dreidimensionales parakristallines Raumgitter, aufgebaut aus den 3 verschiedenen dicken Fadenarten (d 1, d2, d4). Die Abstände zwischen den Fäden werden als Gitterkonstanten (a 1, a2, a3, a4) dieses Raumgitters aufgefaßt. Eine Elementarzelle des Gitters scheint aus einem Geflecht aus d 1- und d 2-Fäden zu bestehen und aus vier darüberliegenden Schichten paralleler d 4-Fäden.

Im 2. Teil der Arbeitshypothese wird auf Grund von Volumenabschätzungen angenommen: die d 1-Fäden des Raumgitters enthalten Rhodopsin, die d 2-Fäden Protein, das nicht Rhodopsin ist, und die (d 4-Fäden enthalten Lipide.

Die Arbeitshypothese wird durch experimentelle Befunde anderer Autoren gestützt, die mit den Methoden der negativen Kontrastierung, der Licht- und Röntgenstrahl-Kleinwinkel-Beugung experimentierten.

Es wird versucht, für einige elektronenmikroskopische Aufnahmen von Dünnschnitten und Gefrierätzabdrucken eine gemeinsame Deutung zu geben (Rosenkranz et al., 1969; Rosenkranz, 1969a).

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Authors and Affiliations

  1. Institut für Zoologie der Technischen Hochschule, Aachen

    Jürgen Rosenkranz

  2. Institut für Zellmorphologie der Ruhr-Universität, Bochum, Germany

    Jürgen Rosenkranz

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  1. Jürgen Rosenkranz
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I wish to thank Prof. Dr. H. Stieve for the interest he took in this work through critical discussions and financial support. I also wish to thank Prof. A. Ruthmann, Ph. D., for introducing me to electron microscopy and for his linguistic aid. That Prof. Dr. K. Mühlethaler, ETH Zürich, and Prof. Dr. F. Schwanitz, KFA Jülich, put their freeze-etching apparatus and electron microscope at my disposal is gratefully acknowledged. The technical assistance of Miss M. Deichmann is also acknowledged.

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Rosenkranz, J. On the fine structure of the frog's rod outer segments, observed by the freeze-etching technique. Z. Zellforsch. 111, 228–262 (1970). https://doi.org/10.1007/BF00339787

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