Super-resolved 3D tracking of cargo transport through nuclear pore complexes

Chowdhury, Rajdeep; Sau, Abhishek; Musser, Siegfried M.

doi:10.1038/s41556-021-00815-6

Cited by 32 publications

(28 citation statements)

References 84 publications

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“…To avoid difficulties due to this issue, we focused on analyzing the distance between neighboring Nup96 subunits, rather than on the nuclear pore sizes. As expected from literature [35], they appeared to be ~38 nm apart (Supp. Fig.…”

Section: Resultssupporting

confidence: 87%

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Saal

Shaib

Mougios

et al. 2022

Preprint

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Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

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Section: Resultssupporting

confidence: 87%

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Saal

Shaib

Mougios

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…To avoid di culties due to this issue, we focused on analyzing the distance between neighboring Nup96 subunits, rather than on the nuclear pore sizes. As expected from literature [35], they appeared to be ~ 38 nm apart (Supplementary Figure S3C).…”

Section: X10ht Allows the Analysis Of Thick Tissuessupporting

confidence: 86%

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Saal

Shaib

Mougios

et al. 2022

Preprint

View full text Add to dashboard Cite

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6–8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

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“…In contrast to essential yeast FG-Nups that have a bimodal (cohesive and FG-rich N-terminal domain vs. highly charged and FG-devoid C-terminal domain) structure such as Nup100, Nup116, and Nup145N, the highly charged extended domain of Nsp1 contains a large number of FG-motifs. Changes in conformation of such FG-Nups with increasing Kap95 concentrations may be more pronounced than for the Nsp1 protein studied here, and as a consequence, such conformational change may lead to changes in the spatial distribution of Kap95 in such systems as well [78,79]. The exact spatial localization of Kap95 molecules within the FG-mesh may, in fact, differ when considering the NPC in its entirety.…”

Section: Consequences Of These Findings For Existing Models Of Nuclea...mentioning

confidence: 80%

Transport receptor occupancy in nuclear pore complex mimics

et al. 2022

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Nuclear pore complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG-Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess “Kap-centric” NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

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Super-resolved 3D tracking of cargo transport through nuclear pore complexes

Cited by 32 publications

References 84 publications

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Transport receptor occupancy in nuclear pore complex mimics

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