THE MACRONUCLEAR ENVELOPE OF TETRAHYMENA PYRIFORMIS GL IN DIFFERENT PHYSIOLOGICAL STATES

Abstract

The pore complexes of the nuclear envelope presumably regulate macromolecular exchange between the cytoplasm and the karyoplasm (7-9, 11, 14-16, 18, 22, 24-27, 30) . We have previously presented quantitative data, obtained by negative staining (29, 32) and thin-sectioning, (31) on the number, size, and fine structure of nuclear pores in the macronuclear envelope of the ciliate protozoon Tetrahymena pyriformis GL in different physiological states . We now relate these results to those obtained by freeze-etch electron microscopy, a technique which allows observation of large areas of partially hydrated biomembranes . We also describe the influence of glutaraldehyde prefixation on the freeze-etch appearance of nuclear pores . MATERIAL AND METHODS CulturesStock cultures of Tetrahymena pyriformis (amicronucleate strain GL) were maintained axenically at 15'C in 2% proteose peptone containing 0 .47 7 0 liver extract . We have examined cells of cultures in the logarithmic, as well as in the stationary, growth phase, and also those of heat-shock synchronized cultures at the end of the synchronization treatment (EHT) and at the division maximum (29, 30) . 2Freeze-Etching and Electron Microscopy Cells in the four different physiological states were centrifugally concentrated into 2 ml of culture medium. Fixation, when performed, was done by addition of 2 ml of 3-4% glutaraldehyde, in 0 .1 M cacodylate (pH 7 .2) .After 15 min the cells were thoroughly washed with buffer, and transferred through buffered solutions of increasing glycerol concentration to a level of 20 0]0 glycerol. Unfixed cells were directly incubated in the glycerol solutions for 1-3 hr. Small samples of packed Tetrahymena cells were frozen on gold-alloy specimen holders in Freon 22 (E . I . duPont de Nemours & Co ., Inc., Wilmington, Del.) . The frozen specimens were replicated according to Moor and Mühlethaler (21) on a Balzers model BA 360 M freeze-etch device (Balzers A . G., Liechtenstein) . The etching time was usually 1 min at -100°C. In some experiments, etching was minimized (see reference 28) by keeping the temperature of the specimen holder at -150°C and by replicating the freshly fractured surface in less than 5 sec after fracturing . Replicas were examined in a Siemens Elmiskop IA, the magnification of which was routinely calibrated by using a carbon grating replica . MeasurementsAll measurements were made on calibrated positives . Pores per square micron were determined by direct counts on fracture faces with large areas of membrane. 100 pores of different macronuclei from

Experimental disintegration of the nuclear envelope. Evidence for pore-connecting fibrils.

“…Therefore, the remaining alternatives that seem plausible to us are that the pore-connecting fibrils represent either (a) skeletal structures which in vivo are apposed to the inner nuclear membrane in a way equivalent to, albeit much thinner than, the "honeycomb layer" or the "fibrous lamina" described in some cell types (2; for references, see introductory paragraph), or (b) intrinsic membranous structures that are exposed after, or rearranged during, the disintegration and the removal of other membrane moieties. From our observation, we favor the second interpretation, which also finds some support in the descriptions of intramembranous threads that connect adjacent pores as seen in tangential sections and freeze-cleavage preparations (23,39,60; for reviews, see 21,25). This interpretation is also supported by the occurrence of carbohydrates with a typical membrane-like pattern in such residual thread material (the possible alternative that the nonmembranous structures of the pore complexes contain carbohydrates of the same composition as ER-membranes seems very unlikely to us, especially since such carbohydrates were not found in high salt extracts from isolated nuclear envelopes and nuclei; for references, see 21, footnote 1) as well as by the finding that some of the membrane cytochromes, particularly cytochrome b6, are retained in detergent-treated nuclei and nuclear membrane preparations (Jarasch and Franke, unpublished data; for discussion and references, see also 21,37,65).…”

Plugs in nuclear pores: Transcripts in early oocyte development identified with nanotechniques

“…This apparent discrepancy is most likely due to technical reasons [Speth and Wunderlich, 1970]. In fact, the fragments used for the EM studies may not be representative for the whole NE.…”

Nuclear envelopes: Structure and biochemistry of the nuclear envelope

“…This may indicate that some shrinkage occurs during dehydration and isolation (compare, for example, Branton & Moor 1964;Franke 1966Franke etal. 1970;Speth & W underlich 1970; Kartenbec This is not observed, however, in some nuclei such as in the am phibian oocyte (Kartenbeck et al 1971;Scheer 1973). There have been, and still are, discussions as to whether the true perimeter of the pore is circular or polygonal (pro circularity: Franke 1966Franke , 1967Franke & Scheer 1970a;Roberts & Northcote 1970pro polygonality: Gall 1967;Kessel 1969;Abelson & Smith 1970;M aul 1971).…”