Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells

Abstract

During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…Our study, revealing the early recruitment of Nup107 to the reforming NE in late anaphase, the slightly delayed recruitment of ketel GFP and WGA-Nup, and the late recruitment of Mtor, is consistent with previous studies in other organisms (Bodoor et al, 1999;Burke and Ellenberg, 2002;Dultz et al, 2008). Along with data recently gathered in human cells (Dultz et al, 2008), it further indicates that NPC reassembly is not simply reversing the sequence of its disassembly. …”

“…Although Nup58 forms part of the central channel of the NPC in mammals (Guan et al, 1995), a previous electron microscopy study suggested that the release of the central transporter or central material occurs at an early stage of NPC disassembly in Drosophila embryos (Kiseleva et al, 2001). Our unexpected result was however corroborated by a study that came out while this manuscript was under revision, revealing that human Nup58 remains longer than Nup133 (another constituent of the Nup107 complex) in fragments of the NE during disassembly (Dultz et al, 2008). During the closed mitosis of the fungus Aspergillus nidulans, however, FG-nups, including those making up the central channel, disperse throughout the cell, whereas constituents of the Nup107 subcomplex persist at NPCs (De Souza et al, 2004;Osmani et al, 2006).…”

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import

“…Both associate with protein phosphatase complexes (18,19), and both are highly mobile nuclear pore proteins (7). They are also both recruited relatively early during the process of post-mitotic NPC formation (20). Some of the assays underlying these observations also point to distinctions between the two nucleoporins, as their off-rates from the pore are different, and their recruitment to newly forming pores is not in precise synchrony (7,20).…”

“…They are also both recruited relatively early during the process of post-mitotic NPC formation (20). Some of the assays underlying these observations also point to distinctions between the two nucleoporins, as their off-rates from the pore are different, and their recruitment to newly forming pores is not in precise synchrony (7,20). Thus, although Nup50 and Nup153 overlap spatially and participate in similar processes (likely in concert), a fundamental question that has not been addressed is whether this partnership is limited to a subset of Nup50 found in the cell.…”

Assembly of nuclear pore complexes mediated by major vault protein

“…Nup62 is a member of the FG-repeat containing nucleoporins; it localizes to the central NPC channel and marks the formation of completed NPCs (Dultz et al, 2008). In addition, the functional status of the newly formed NPCs was examined by scoring the nuclear uptake of the nucleolar protein fibrillarin.…”