A Comparative Study of Nucleocytoplasmic Interactions

Feldherr, Carl M.

doi:10.1083/jcb.42.3.841

Cited by 32 publications

(14 citation statements)

References 14 publications

(16 reference statements)

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“…[4][5][6][7][8][9], and one often observes particles about 70-160 A in diameter arranged peripherally in a ring . They are occasionally arrayed in sets cf eight within a distinct line surrounding the pore (Figs .…”

Section: Resultsmentioning

confidence: 99%

THE MACRONUCLEAR ENVELOPE OF TETRAHYMENA PYRIFORMIS GL IN DIFFERENT PHYSIOLOGICAL STATES

Speth¹,

Wunderlich²

1970

The Journal of Cell Biology

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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

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Section: Resultsmentioning

confidence: 99%

THE MACRONUCLEAR ENVELOPE OF TETRAHYMENA PYRIFORMIS GL IN DIFFERENT PHYSIOLOGICAL STATES

Speth¹,

Wunderlich²

1970

The Journal of Cell Biology

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“…Independently, pulse chase experiments combined with subcellular fractionation, as well as nuclear transplantation experiments, have revealed the transfer of proteins from the cytoplasm to the nucleus in HeLa cells (Byers, Platt, & Goldstein, 1963;Speer & Zimmerman, 1968). Other early transport studies were based on thin-section EM observations or localization of radiolabeled molecules by autoradiography following microinjections, notably into Xenopus oocytes (Feldherr, 1965(Feldherr, , 1969. Other early transport studies were based on thin-section EM observations or localization of radiolabeled molecules by autoradiography following microinjections, notably into Xenopus oocytes (Feldherr, 1965(Feldherr, , 1969.…”

Section: Nups Are Composed Of a Limited Set Of Structural Domainsmentioning

confidence: 99%

Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies

Floch¹,

Palancade²,

Doye³

2014

Methods in Cell Biology

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“…Store in the dark at 4°C (see Note 4). 7. Check the quality of the protein-coated gold particles by negative-staining EM.…”

Section: Conjugation Of Proteins With Colloidal Goldmentioning

confidence: 99%

Use of Intact Xenopus Oocytes in Nucleocytoplasmic Transport Studies

Panté

2006

Xenopus Protocols

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SummaryBecause of its large nucleus, the Xenopus laevis oocyte offers an excellent system to study nucleocytoplasmic transport. This system, in combination with electron microscopy, has provided much of our insight into the mechanisms of nuclear import and export. In a typical experiment, the nuclear transport substrate is first labeled with colloidal gold, and the resulting complex is injected into the cytoplasm (to study nuclear import) or the nucleus (to study nuclear export) of Xenopus oocytes. The oocytes are then fixed, dehydrated, infiltrated, and embedded into an epoxy resin. Following resin polymerization, thin sections of oocyte nuclei are obtained and examined under an electron microscope. Subsequent evaluation of the position and distribution of the gold-labeled substrate reveals whether the substrate has undergone nuclear import (or export) and the position of ratelimiting events. This chapter describes in detail the protocols for performing electron microscopy import assays with Xenopus oocytes and presents some data illustrating the types of experiments possible using this system.

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A Comparative Study of Nucleocytoplasmic Interactions

Cited by 32 publications

References 14 publications

THE MACRONUCLEAR ENVELOPE OF TETRAHYMENA PYRIFORMIS GL IN DIFFERENT PHYSIOLOGICAL STATES

THE MACRONUCLEAR ENVELOPE OF TETRAHYMENA PYRIFORMIS GL IN DIFFERENT PHYSIOLOGICAL STATES

Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies

Use of Intact Xenopus Oocytes in Nucleocytoplasmic Transport Studies

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