The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Walther, Tobias C.; Pickersgill, Helen; Cordes, Volker C.; Goldberg, Martin W.; Allen, Terry D.; Mattaj, Iain W.; Fornerod, Maarten

doi:10.1083/jcb.200202088

Cited by 187 publications

(213 citation statements)

References 95 publications

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“…Equivalent accumulation was seen, however, in the presence of an independent antibody specific for Nup358 that was active in perturbing progress of nuclear envelope breakdown. Our conclusion that Nup358 activity can be blocked without significantly altering import through the pore is consistent with reports in the literature in which this nucleoporin was depleted by various means without impacting nuclear import (Walther et al, 2002;Salina et al, 2003, Forler et al, 2004, Bernad et al, 2004. Nup358 has been suggested to play a supporting role in exportin-1 (CRM1)-dependent export (Bernad et al, 2004); however, this is unlikely to contribute to inhibition of nuclear disassembly as a potential decrease in nuclear export sequence (NES) export would be predicted to increase nuclear levels of cyclin B and speed, rather than delay, mitotic entry (Yang et al, 1998).…”

Section: Discussionsupporting

confidence: 92%

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Prunuske

Liu

Elgort

et al. 2006

MBoC

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When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated. INTRODUCTIONThe nuclear envelope creates a barrier that is critical to maintenance of the environment in the nucleus, specialized to support transcription and DNA replication. This double lipid membrane bilayer consists of an outer nuclear membrane, which is continuous with the endoplasmic reticulum (ER), and an inner nuclear membrane, which contains at least 78 different proteins, anchored via protein-protein interactions to the underlying nuclear lamina and chromatin . The nuclear envelope is perforated by nuclear pores, through which communication between the cytoplasm and nucleus occurs (Suntharalingam and Wente, 2003;Weis, 2003;Fahrenkrog et al., 2004;Pante, 2004). Despite the large size of the macromolecular nuclear pore complex (125 MDa in vertebrates), it is comprised of only ϳ30 different proteins, or nucleoporins (Nups), each present in multiple copies (Rout et al., 2000;Cronshaw et al., 2002). In metazoan cells, all these components-the lamina, nuclear pores, as well as the ...

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Section: Discussionsupporting

confidence: 92%

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Prunuske

Liu

Elgort

et al. 2006

MBoC

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“…Membrane vesicles were bound to the chromatin surface, but they did not fuse to form a closed NE and no NPC assembly was detected either by immunofluorescence or electron microscopy. Of the nucleoporins thus far tested, depletion of only three have produced a similar defect in NE formation (Finlay et al, 1991;Powers et al, 1995;Grandi et al, 1997;Walther et al, 2001Walther et al, , 2002. Two nucleoporins are the integral pore membrane proteins NDC1 and POM121 Mansfeld et al, 2006) that reside, together with lamin B receptor, in a membrane vesicle population, which has a high avidity for the chromatin surface Ulbert et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Hawryluk-Gara

Platani

Santarella

et al. 2008

MBoC

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Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly. INTRODUCTIONThe nuclear envelope (NE) provides a physical barrier between the nucleus and cytoplasm and their unique metabolic tasks. The NE is defined by three morphologically distinct regions. The outer nuclear membrane (ONM) is continuous with the rough endoplasmic reticulum (ER) and contains a similar set of proteins (reviewed in Mattaj, 2004). The inner nuclear membrane (INM) lies adjacent to the nucleoplasm and contains a unique repertoire of proteins that, in part, mediate its interactions with the nuclear lamina and chromatin. Finally, at numerous locations along the NE, the ONM and INM are interrupted by pores where the INM and the ONM are bridged by a connecting membrane termed the pore membrane (POM) domain. Within these pores reside the nuclear pore complexes (NPCs), aqueous channels that provide portals for both passive and active transport of macromolecules.An NPC contains ϳ30 nucleoporins (Nups), many of which are evolutionarily conserved in structure and function (Tran and Wente, 2006). In many cases these Nups are organized into subcomplexes that are present in multiple copies, and they are distributed around the central axis of the NPC, contributing to its characteristic eightfold symmetry. Different groups of Nups and their respective subcomplexes contribute to distinct structural components of the NPC. For example, Nup214/Nup84 are components of the cytoplasmic fibrils (Kraemer et al., 1994;Bastos et al., 1997), the Nup53/Nup155 complex is part of the central core (Marelli et al., 1998), and Nup153 and Tpr contribute to the nucleoplasmic ring and fibrils (Krull et al., 2004).When metazoan cells enter mitosis, their NE is disassembled allowing for the mitotic spindle to access the condensed chromatin. Several models based on previous findings have been proposed to explain the mechanism by which this occurs (reviewed in Hetzer et al., 2005). NE disassembly is accompanied by the phosphorylation of multiple NE proteins including the lamins and several Nups (Burke and Ellenberg, 2002, and refer...

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“…While this approach provides some measure of the position of the NPC within the plane of the NE, it does not provide information on the position of the NPC structure along the normal to the NE. Based on electron microscopy data [10,37], we have assumed that the path of the NE in equatorial nuclear cross-sections passes through the center of NPCs. The position of the NE was determined by bright-field microscopy as follows.…”

Section: Nuclear Envelope Localizationmentioning

confidence: 99%

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Yang

Musser

2006

Methods

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The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

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The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Cited by 187 publications

References 95 publications

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

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