Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event

Christiane Wiese, Martin W. Goldberg, Terence D. Allen, Katherine Lee Wilson

Research output: Contribution to journalArticle

Abstract

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Original languageEnglish (US)
Pages (from-to)1489-1502
Number of pages14
JournalJournal of Cell Science
Volume110
Issue number13
StatePublished - 1997

Fingerprint

Nuclear Envelope
Xenopus
Chromatin
Lamins
Membranes
Ribosomes
Nuclear Pore Complex Proteins
Nuclear Pore
Porins
Wheat Germ Agglutinins
Cell Nucleus Active Transport
Active Biological Transport
Endoplasmic Reticulum
Electron Scanning Microscopy
Cytosol
Lenses
Ovum
Membrane Proteins
Electrons
Proteins

Keywords

  • FEISEM
  • Mitosis
  • Nucleocytoplasmic transport
  • Nucleoporin

ASJC Scopus subject areas

  • Cell Biology

Cite this

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event. / Wiese, Christiane; Goldberg, Martin W.; Allen, Terence D.; Wilson, Katherine Lee.

In: Journal of Cell Science, Vol. 110, No. 13, 1997, p. 1489-1502.

Research output: Contribution to journalArticle

@article{e07c9f7e492b4a54945ffeffc7eef23d,
title = "Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event",
abstract = "We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.",
keywords = "FEISEM, Mitosis, Nucleocytoplasmic transport, Nucleoporin",
author = "Christiane Wiese and Goldberg, {Martin W.} and Allen, {Terence D.} and Wilson, {Katherine Lee}",
year = "1997",
language = "English (US)",
volume = "110",
pages = "1489--1502",
journal = "Journal of Cell Science",
issn = "0021-9533",
publisher = "Company of Biologists Ltd",
number = "13",

}

TY - JOUR

T1 - Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event

AU - Wiese, Christiane

AU - Goldberg, Martin W.

AU - Allen, Terence D.

AU - Wilson, Katherine Lee

PY - 1997

Y1 - 1997

N2 - We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

AB - We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

KW - FEISEM

KW - Mitosis

KW - Nucleocytoplasmic transport

KW - Nucleoporin

UR - http://www.scopus.com/inward/record.url?scp=0030794456&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030794456&partnerID=8YFLogxK

M3 - Article

VL - 110

SP - 1489

EP - 1502

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 13

ER -