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© The Rockefeller University Press, 0021-9525/1997//1193 $5.00
The Journal of Cell Biology, Volume 138, Number 6, , 1997 1193-1206

Article

Nuclear Membrane Dynamics and Reassembly in Living Cells: Targeting of an Inner Nuclear Membrane Protein in Interphase and Mitosis



Jan Ellenberg*, Eric D. Siggia{ddagger}, Jorge E. Moreira*, Carolyn L. Smith§, John F. Presley*, Howard J. Worman||, and Jennifer Lippincott-Schwartz*

* Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health (NIH), Bethesda, Maryland 20892; {ddagger} Department of Physics, Cornell University, Ithaca, New York 14853; § Light Imaging Facility, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland 20892; and || Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York 10032

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR–GFP). Photobleaching techniques revealed the majority of LBR–GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR–GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0.41 ± 0.1 µm2/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR–GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR–GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR–GFP in ER elements at contact sites with chromatin. LBR–GFP–containing ER membranes then wrapped around chromatin over the course of 2–3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30–80 min. Thus, selective changes in lateral mobility of LBR–GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.

Abbreviations used in this paper: FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; LBR, lamin B receptor; NE, nuclear envelope.

Address all correspondence to Jennifer Lippincott-Schwartz, Cell Biology and Metabolism Branch, NICHD, NIH, Bethesda, MD 20892. Tel.: (301) 402-1010. Fax: (301) 402-0078. e-mail: jlippin{at}helix.nih.gov

J. Ellenberg was supported by a predoctoral fellowship of the Boehringer Ingelheim Fonds, Germany. H.J. Worman is an Irma T. Hirschl Scholar and supported by grants from the National Institutes of Health (RO1-CA66974) and the American Cancer Society (RPG-94-006-03-CB).


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