Nuclear pore complexes (NPCs) mediate all traffic of macromolecules across the nuclear envelope (NE). They are large protein assemblies composed of multiple copies of ~30 different proteins, the nucleoporins (Nups), which are organized in about 10 subcomplexes and arranged with eightfold symmetry. In metazoa, NPCs are stable throughout interphase (Daigle et al., 2001
) but disassemble into their subcomplexes during mitosis. When the NE breaks down in pro/metaphase, most Nups become cytoplasmic and transmembrane Nups relocalize to the ER together with other nuclear membrane proteins (Ellenberg et al., 1997
; Yang et al., 1997
; Daigle et al., 2001
; Beaudouin et al., 2002
). Reassembly occurs during anaphase and telophase when the NE is rebuilt around chromatin.
In live cells, NE disassembly has been shown to start by partial disassembly of NPCs, with Nup98 leaving the NE early followed by dissociation of Nup153 and Nup214 before the NE is completely permeabilized. The membrane Nup POM121 dissociates from NE fragments only after permeabilization (Beaudouin et al., 2002
; Lenart et al., 2003
). In fixed cells, the nuclear basket Nup Tpr dissociates from the NE before Nup107 but later than Nup98 and Nup50 (Hase and Cordes, 2003
More is known about the mechanism of postmitotic NPC assembly. In vitro studies of nuclear assembly in Xenopus laevis
egg extracts have shed light on the essential role of the Ran–importin system, which regulates the release of several Nups from importin in proximity to chromatin, enabling them to reassociate and form NPCs (Harel et al., 2003a
; Walther et al., 2003b
). Several Nups bind to chromatin in early anaphase before membrane association (Belgareh et al., 2001
; Walther et al., 2003a
), where they have been postulated to form a prepore (Suntharalingam and Wente, 2003
; Wozniak and Clarke, 2003
; Rabut et al., 2004b
). The mechanism of subsequent insertion into the membrane and full assembly of the NPC remains to be understood.
For some Nups, the order of reassociation with the reforming NE was investigated in various experimental systems, fixed cells of different mammalian species, or nuclei assembled in X
egg extracts. Together, these data predict that the Nup107–160 complex, Nup153, Nup98, and POM121 bind during anaphase, followed by the Nup62 and Nup93 complexes, Nup358, and Nup214 in telophase, whereas Tpr and gp210 reassemble only in early G1 (for review see Burke and Ellenberg, 2002
Evidence for structural disassembly and reassembly intermediates has been provided by field emission scanning electron microscopy. Porelike structures of different levels of complexity could be visualized in egg extract nuclei (Goldberg et al., 1997
; Wiese et al., 1997
; Kiseleva et al., 2001
) and a rough time course of the formation of these structures could be established in Drosophila melanogaster
embryos (Kiseleva et al., 2001
). Their protein composition remained, however, unclear.
Our current knowledge predicts that NPC disassembly and reassembly are ordered processes that proceed via a defined set of intermediates formed by sequential interactions of NPC subcomplexes. However, the precise order in which the different subcomplexes bind, the kinetics of the assembly events, and the functional state of the different intermediates are unknown. To address this, we systematically investigated the kinetics of mitotic NPC disassembly and reassembly by time lapse confocal microscopy in single dividing cells. Simultaneously, we monitored import competence of the nucleus. We analyzed a set of GFP-tagged Nups (Rabut et al., 2004a
) representing eight different NPC subcomplexes. Our results show that NPC assembly is indeed a highly ordered process that proceeds in a stepwise fashion. Partially assembled NPCs were already import competent, which indicates that several Nups may not be required to reestablish import function. Regarding NPC disassembly, we found it to occur more rapidly than assembly and not simply in the reverse order, which could indicate a distinct mechanism. Based on our data, we present the first comprehensive model for the order, composition, and functional state of NPC disassembly and reassembly intermediates in living cells.