Cryo-electron Tomography Provides Novel Insights into Nuclear Pore Architecture: Implications for Nucleocytoplasmic Transport

Stoffler, Daniel; Feja, Bernhard; Fahrenkrog, Birthe; Walz, Jochen; Typke, Dieter; Aebi, Ueli

doi:10.1016/s0022-2836(03)00266-3

Cited by 222 publications

(257 citation statements)

References 47 publications

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“…Flexible filaments extend ∼50 nm into the cytoplasm, and a filamentous open basket structure extends ∼75 nm into the nucleoplasm (Fig. 1) [1,4]. High localization precision for cargos moving within these NPC structures (spanning ∼200 nm) is most easily obtained from micrometer-scale images using particle tracking algorithms.…”

Section: Visualizing Single Cargo Molecules Interacting With Nuclear mentioning

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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Section: Visualizing Single Cargo Molecules Interacting With Nuclear mentioning

confidence: 99%

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

Yang

Musser

2006

Methods

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“…It has been demonstrated that NPCs can undergo massive structural changes induced by cellular signals such as calcium or nucleotides (Jarnik and Aebi 1991;Stoffler et al 1999bStoffler et al , 2003Fahrenkrog and Aebi 2003), suggesting that the NPC as a whole is extremely sensitive to environmental conditions and can exhibit significant structural plasticity, which in turn may be crucial for efficient cargo translocation through its central pore. Hence, a mechanistic understanding of nucleocytoplasmic transport at the level of the response of individual NPCs requires that these distinct functional states be visualized, characterized, and identified.…”

Section: Imaging the Nuclear Pore Complexmentioning

confidence: 99%

“…a, c and d reproduced with permission from Nature Reviews Molecular Cell Biology 4:757-766 copyright (2003) Macmillan Magazines Ltd. b reprinted from Stoffler et al J Mol Biol 328:119-130 (2003) with permission from Elsevier. e and f reprinted from Stoffler et al Curr Opin Cell Biol 11:391-401 (1999) with permission from Elsevier Stoffler et al 1999aStoffler et al , 2003Beck et al 2004).…”

Section: Imaging the Nuclear Pore Complexmentioning

confidence: 99%

“…The central pore often appears plugged with a 'particle' of variable size and appearance, termed central 'plug' or 'transporter' (Akey 1990;Reichelt et al 1990;Jarnik and Aebi 1991;Akey and Radermacher 1993;Kiseleva et al 1998). The functional role of the plug remains to be established, although it is possible for this polymorphic structure to consist of a combination of dynamically interacting phenylalanineglycine (FG)-domains and cargo being caught in transit, rather than representing a stationary component of the NPC (Jarnik and Aebi 1991;Stoffler et al 1999bStoffler et al , 2003.…”

Section: Imaging the Nuclear Pore Complexmentioning

confidence: 99%

“…These stereo images, which represent some of the most spectacular and informative electron micrographs found in literature to this day, have started a new chapter in the history of NPC structure. In fact, these remarkable ultrastructural data have challenged many researchers in the field-including ourselves-to replicate, scrutinize, and elaborate on these novel views of NPC architecture Akey 1990;Jarnik and Aebi 1991;Goldberg et al 1997;Kiseleva et al 1998;Stoffler et al 2003;Beck et al 2004). Last but not least, Ris' morphological data also had a profound impact on the molecular dissection of NPC structure (Pante and Aebi 1993;Pante et al 1994;Fahrenkrog et al 1998;Rout et al 2000;Paulillo et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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From the trap to the basket: getting to the bottom of the nuclear pore complex

2006

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Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the doublemembraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of highresolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.

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Overview of Traditional Experimental Arsenal to Study Biomolecular Structure and Dynamics

Kaltashov

Eyles

2012

Mass Spectrometry in Structural Biology and Biophysics

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Cryo-electron Tomography Provides Novel Insights into Nuclear Pore Architecture: Implications for Nucleocytoplasmic Transport

Cited by 222 publications

References 47 publications

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

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

From the trap to the basket: getting to the bottom of the nuclear pore complex

Overview of Traditional Experimental Arsenal to Study Biomolecular Structure and Dynamics

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