Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan, Jervis Vermal; Kahnwald, Maurice; Cieslinski, Konstanty; Hoess, Philipp; Peneti, Sudheer Kumar; Reitberger, Manuel; Heid, Daniel; Kasuba, Krishna Chaitanya; Hörner, Sarah Janice; Li, Yiming; Wu, Yule; Mund, Markus; Matti, Ulf; Pereira, Pedro M.; Henriques, Ricardo; Nijmeijer, Bianca; Kueblbeck, Moritz; Sabinina, Vilma Jimenez; Ellenberg, Jan; Ries, Jonas

doi:10.1038/s41592-019-0574-9

Cited by 287 publications

(332 citation statements)

References 68 publications

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“…Furthermore, we imaged a HaloTag fusion of Nup96 32 , a protein of the nuclear pore complex. Nuclear pores possess a regular circular shape with an internal diameter of about 100 nm 33,34 .…”

Section: Resultsmentioning

confidence: 99%

Photoactivation of silicon rhodamines via a light-induced protonation

et al. 2019

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Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

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Section: Resultsmentioning

confidence: 99%

Photoactivation of silicon rhodamines via a light-induced protonation

et al. 2019

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“…4). O-GlcNAc was concentrated the most at the center and lower at the periphery, resembling the binding pattern of WGA 33,34 . In the side view of the NPC, Nup96 was situated along two lines delineating the top and bottom of the symmetric core of the NPC, separated by ∼50 nm (Fig.…”

Section: Main Textmentioning

confidence: 70%

“…To gain insight into how O-GlcNAcylation alters the NPC, we investigated the distribution of O-GlcNAc within the NPC. Previous studies showed that wheat germ agglutinin (WGA), a GlcNAc-binding lectin, is predominantly localized to the center of the NPC 33,34 . However, WGA may misrepresent the O-GlcNAc distribution due to its multiple binding sites and considerable size (36 kDa) 35 .…”

Section: Main Textmentioning

confidence: 99%

O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

Yoo

Mitchison

2020

Preprint

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Macromolecular transport across the nuclear envelope is fundamental to eukaryotic cells and depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs)1,2. The NPC is enriched in O-linked N-acetylglucosamine (O-GlcNAc) modification3-8, but its functional role in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells and showed that the O-GlcNAc modification of the NPC accelerates the nucleocytoplasmic transport in both directions. Super-resolution imaging of O-GlcNAc revealed strong enrichment at the FG barrier of the NPC channel. O-GlcNAc modification also promoted the passive permeation of a small, inert protein through NPCs. Our results suggest that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier.

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“…Nucleoporin, the constituent building blocks of the nuclear pore complex (NPC), are heavily O-GlcNAcylated and can be stained by the O-GlcNAc-recognizing lectin wheat germ agglutinin (WGA), which have been utilized for super-resolution imaging of the central channel of NPC 13 . Heavy GalNAz labeling was co-localized with NPC staining on the nuclear envelop ( Supplementary Fig.…”

Section: Fig1 | Click-exm Imaging Of Lipidsmentioning

confidence: 99%

Click-ExM enables expansion microscopy for all biomolecules

Sun

Fan

Zhang

et al. 2020

Preprint

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Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids.Here we develop click-ExM, which integrates click-labeling into ExM to enable a "one-stop-shop" method for nanoscale imaging of various types of biomolecules.Using 18 clickable labels for click-ExM imaging of DNA, RNA, proteins, lipids, glycans and small molecules, we demonstrate its universality, compatibility with signal-amplification techniques, and broad applications in cellular imaging.By physically expanding proteins or RNA of fixed specimens embedded in a swellable polymer hydrogel, expansion microscopy (ExM) enables nanoscale imaging by using conventional diffraction-limited microscopes 1 . As a critical step of ExM, the biomolecules or the labeled fluorophores need to be covalently anchored into the polymer network and preserved during homogenization of the fixed cells (e.g., by strong protease digestion) to ensure isotropic expansion. This imposes a great challenge to develop tailored protocols for specific imaging targets including proteins and RNA; however, other types of biomolecules such as lipids, glycans, and small molecules are currently not amenable to ExM.To expand the applicability of ExM, we developed click-ExM, a variant of ExM, into which a unified method to label, anchor, and preserve fluorescent signals for all kinds of biomolecules is integrated (Fig. 1a). To develop such a unified protocol, we exploited click-labeling, which has emerged as a versatile tool for fluorescence imaging

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Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Cited by 287 publications

References 68 publications

Photoactivation of silicon rhodamines via a light-induced protonation

Photoactivation of silicon rhodamines via a light-induced protonation

O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

Click-ExM enables expansion microscopy for all biomolecules

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