Nucleocytoplasmic transport: taking an inventory

Fried, Howard M.; Kutay, Ulrike

doi:10.1007/s00018-003-3070-3

Cited by 457 publications

(438 citation statements)

References 298 publications

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“…2A-2D) interferon-c expectedly had no influence on the distribution of either antibody. However, IFN-c treatment of cells triggers the association of both STAT1 and importin-b with the karyopherin importin-a, which affords their nuclear import via a GTPase-dependent mechanism (3,30,31). Surprisingly, while the translocating antibody accumulated in the nucleus, it nonetheless prevented the concurrent nuclear accumulation of STAT1 (Fig.…”

Section: Characterization Of a Translocating Importin-b Antibodymentioning

confidence: 95%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-b antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-a, but by diverse mechanisms. The translocating antibody blocked the association with importin-a, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-a binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives. ' 2008 International Society for Advancement of Cytometry Key terms antibody; native protein; microinjection; nucleocytoplasmic shuttling; import; export; GFP TRANSPORT of proteins and macromolecules between the nucleus and the cytoplasm of eukaryotic cells is an important mechanism for cytoplasmic regulation of nuclear activities. Nucleocytoplasmic exchange occurs through specialized channels called ''nuclear pore complexes'' (NPCs), which perforate the double layer of the nuclear envelope (1). The NPCs function as selectivity filters allowing passive diffusion of molecules up to $40 kDa, whereas components of higher molecular weight are usually restricted from autonomous exchange (2,3). A well-characterized pathway for nuclear transport of proteins relies on the GTPase Ran and the transport factors (also called carriers or karyopherins) importin-a and importin-b or CRM1 for import or export, respectively (4,5). In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or i...

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Section: Characterization Of a Translocating Importin-b Antibodymentioning

confidence: 95%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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“…Larger molecules, up to ∼25 MDa (∼40 nm diameter), must form a complex with at least one transport receptor to transit through the NPC ("carrier-mediated, signal-dependent transport," or "facilitated translocation") [7][8][9]. Importins and exportins are soluble protein cofactors that recognize and bind to import and export cargos through nuclear localization sequences (NLSs) and nuclear export sequences (NESs), respectively.…”

Section: Overview Of Nuclear Transportmentioning

confidence: 99%

“…Import complexes (ICs), consisting of cargo and importin(s), are disassembled after transit through the NPC by Ran-GTP, the GTPbound form of the G-protein Ran. In contrast, export complexes, consisting of cargo, exportin and Ran-GTP, disassemble upon Ran-GTP activation (leading to GTP hydrolysis) by Ran's cytoplasmically localized GTPase activating protein, RanGAP [8]. The Ran-GTP concentration gradient is established and maintained by cytoplasmic RanGAP and nucleoplasmic RanGEF, the chromosome-bound guanine-nucleoside exchange factor for Ran, which catalyzes GDP/GTP exchange ( Fig.…”

Section: Overview Of Nuclear Transportmentioning

confidence: 99%

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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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“…In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.In eukaryotes, the movement of proteins and RNAs between the nucleus and the cytoplasm is an essential cellular process that is mediated by soluble transport receptors (26,52). In addition, the small GTPase Ran (Gsp1 in yeast) and its cytoplasmic and nuclear effectors are needed (12).…”

mentioning

confidence: 99%

Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast

Neuber

Franke

Wittstruck

et al. 2008

Molecular and Cellular Biology

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The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the ␥-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.In eukaryotes, the movement of proteins and RNAs between the nucleus and the cytoplasm is an essential cellular process that is mediated by soluble transport receptors (26,52). In addition, the small GTPase Ran (Gsp1 in yeast) and its cytoplasmic and nuclear effectors are needed (12). Transport receptors involved in nuclear import reactions are usually termed importins, and they recognize their cargoes via a specific amino acid motif, the nuclear localization sequence, or NLS. By analogy, nuclear export reactions are mediated by so-called exportins. Xpo1 (also known as Crm1) was one of the first nuclear export receptors to be identified (23,27,50,64) and recognizes a short leucine-rich amino acid sequence on its cargo, called a nuclear export signal, or NES. Numerous export cargoes have now been identified for the mammalian Crm1 protein, including protein kinase A inhibitor PKI, p53, and the human immunodeficiency virus protein Rev (20,25,66). In addition, Crm1 is involved in the nuclear export of several classes of RNAs such as viral pre-mRNAs, ribosomal RNAs, and snRNAs (54). In contrast, few NES-containing proteins have been identified in Saccharomyces cerevisiae. Although the budding yeast Crm1 orthologue Xpo1 is involved in nuclear export of Hog1 (19), Yap1 (76), Ssb1 (62), Ace2 (36), and Prp40 (46), few of the NESs involved have been characterized at a molecular level.Recently, alongside its well-established role in nuclear export, mammalian Crm1 was shown to be involved in the control of centrosome duplication (71). Centrosomes function as microtubule (MT) organizing centers in mammalian somatic cells and direct the formation of a bipolar spindle during mitosis (reviewed in reference 6). In another study, Crm1 was found to bind to kinetochores, complex protein assemblies that are needed to establish the attachment of MTs to chromatin (4). These studies point to an unanticipated role of the exportin Crm1 in control of spin...

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Nucleocytoplasmic transport: taking an inventory

Cited by 457 publications

References 298 publications

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

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

Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast

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