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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic
transport, the mechanism of NPC assembly into pores
formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast
Saccharomyces cerevisiae. The nucleoporin Nup49p
was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP-Nup49p donor cell was
mated with a recipient cell harboring only unlabeled
Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP-Nup49p, we could assess whether NPCs were
able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed
that fluorescent NPCs moved and encircled the entire
nucleus within 25 min after fusion. When assays were
done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP-Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably
due to new NPC biogenesis or to exchange of GFP-
Nup49p into existing recipient NPCs. Interestingly, in a
number of existing mutant strains, NPCs are clustered
together at permissive growth temperatures. This has
been explained with two different hypotheses: by
movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central
location (such as the spindle pole body) with NPCs in
mutant cells unable to move away from this point. Using the GFP-Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC
clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP-
Nup49p-labeled NPCs, assembled at 23°C, moved into
clusters when the cells were shifted to growth at 37°C.
These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to
form NPC clusters in mutant strains. Such clusters may
result by releasing NPCs from a nuclear tether, or by
disappearance of a protein that normally prevents pore
aggregation. This system represents a novel approach
for identifying regulators of NPC assembly and movement in the future.
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