Nuclear envelope assembly in Xenopus extracts ... - Semantic Scholar

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INTRODUCTION. The nuclear envelope consists of the nuclear pore complexes. (NPCs), the nuclear lamina, and the nuclear membranes. (reviewed by ...
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Journal of Cell Science 110, 1489-1502 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS1376

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent ‘envelope smoothing’ event Christiane Wiese1,*, Martin W. Goldberg2, Terence D. Allen2 and Katherine L. Wilson1,† 1Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA 2CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, M20 9BX, UK

*Present address: Carnegie Institute of Washington, 115 W. University Parkway, Baltimore MD, 21210, USA †Author for correspondence (e-mail: [email protected])

SUMMARY 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 chromatinattachment 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.

INTRODUCTION

organized into three distinct domains: the inner and outer nuclear membranes, and the pore membrane domain. Because the outer nuclear membrane functions as part of the rough endoplasmic reticulum (ER), the lumen enclosed by the inner and outer membranes is continuous with the ER lumen. Each membrane domain carries a unique set of proteins: lamina- and chromatin-binding proteins reside on the inner nuclear membrane (reviewed by Gerace and Foisner, 1994), proteins involved in rough ER function (e.g. SRP-receptors and ribosomes) are found on the outer nuclear membrane (Rapoport et al., 1996), and integral membrane NPC proteins, such as gp210 (Gerace et al., 1982) and POM121 (Hallberg et al., 1993), reside in the pore membrane domain. Nuclear envelope assembly has been studied in vitro using nuclear reconstitution systems pioneered by Lohka and Masui (reviewed by Lohka, 1988; Wilson and Wiese, 1996). Extracts made from fractionated Xenopus eggs can assemble structurally and functionally normal nuclei around demembranated Xenopus sperm chromatin: these nuclei replicate their DNA,

The nuclear envelope consists of the nuclear pore complexes (NPCs), the nuclear lamina, and the nuclear membranes (reviewed by Marshall and Wilson, 1997). Molecular transport across the nuclear envelope occurs through the NPCs, which are 125,000 kDa structures positioned in holes or pores where the inner and outer membranes merge (the ‘pore membrane domain’). NPCs allow the free diffusion of small (