The nuclear pore complex: from molecular architecture to functional dynamics

Stoffler, Daniel; Fahrenkrog, Birthe; Aebi, Ueli

doi:10.1016/s0955-0674(99)80055-6

Cited by 318 publications

(290 citation statements)

References 2 publications

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“…The size of the nuclear pore complex (NPC) is B55 Å , or 25-30 nm, 198 in diameter and permits the passage of molecules with a MW of p40 kDa. 199 Larger molecules, however, require a mechanism of active transport in a quiescent cell.…”

Section: Nuclear Entrymentioning

confidence: 99%

Intracellular trafficking of nonviral vectors

2005

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Section: Nuclear Entrymentioning

confidence: 99%

Intracellular trafficking of nonviral vectors

2005

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“…These FG-Nups are distributed throughout the NPC structure on both the cytoplasmic filaments and nuclear basket, and in the pore itself [3,10,11]. The FG-repeat regions are highly unfolded (disordered) and highly flexible [12].…”

Section: Overview Of Nuclear Transportmentioning

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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“…[14][15][16][17][18] In our model of plasmid nuclear import, we propose that transcription factors present in the cytoplasm bind to the DTS and coat the plasmid with nuclear localization sequences (NLSs) that utilize importins to cross the NPC, which regulates the nucleocytoplasmic shuttling of macromolecules during interphase. [19][20][21] We have also developed a tissue-specific DTS, utilizing the smooth muscle g-actin (SMGA) promoter, that mediates nuclear import of pDNA specifically in smooth muscle cells (SMCs). 10 The SMGA DTS was shown to drive SMCspecific plasmid nuclear import in rat mesenteric vessels following electroporation into living animals, proving its viability for in vivo gene transfer.…”

Section: Introductionmentioning

confidence: 99%

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Miller

Dean

2008

Gene Ther

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Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle g-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.

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The nuclear pore complex: from molecular architecture to functional dynamics

Cited by 318 publications

References 2 publications

Intracellular trafficking of nonviral vectors

Intracellular trafficking of nonviral vectors

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

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

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