NUCLEAR-MEMBRANE DYNAMICS AND REASSEMBLY IN LIVING CELLS - TARGETING OF AN INNER NUCLEAR-MEMBRANE PROTEIN IN INTERPHASE AND MITOSIS

The mechanisms of localization and retention of membrane proteins in t he 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 co mpletely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpop ulation of LBR-GFP within ER membranes, by contrast, was entirely mobi le and diffused rapidly and freely (D = 0.41 +/- 0.1 mu m(2)/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR-G FP redistributing into the interconnected ER membrane system in promet aphase, exhibiting the same high mobility and diffusion constant as ob served in interphase ER membranes. LBR-GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the in tegrity of the ER was maintained in mitosis, with little or no fragmen tation and vesiculation. At the end of mitosis, nuclear membrane refor mation coincided with immobilization of LBR-GFP in ER elements at cont act 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 loc alization to the inner nuclear membrane during interphase. Such change s, rather than vesiculation mechanisms, also underlie the redistributi on of this molecule during NE disassembly and reformation in mitosis.