The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ~30 different proteins, termed nucleoporins, whose arrangement within the NPC is a major unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133–Nup107 with their long axes approximately parallel to the nuclear envelope plane. This method can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and could monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines.
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