Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

Göttfert, Fabian; Wurm, Christian A.; Mueller, Veronika; Berning, Sebastian; Cordes, Volker C.; Honigmann, Alf; Hell, Stefan W.

doi:10.1016/j.bpj.2013.05.029

Cited by 273 publications

(232 citation statements)

References 17 publications

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“…To visualize polyadenylated mRNA, fluorescence in situ hybridization was carried out with a Cy3-labeled (dT) 40 probe. U2OS cells were seeded on coverslips in six-well plates, treated with PR 20 for 8 h, and fixed with 4% (wt/vol) paraformaldehyde in PBS.…”

Section: Methodsmentioning

confidence: 99%

Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Shi

Mori

Nizami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

222

191

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The toxic proline:arginine (PR n ) poly-dipeptide encoded by the (GGGGCC) n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PR n poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PR n poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PR n -mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PR n poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PR n poly-dipeptide toxicity in the context of a prominent form of ALS.C9orf72 repeat expansion | PR n poly-dipeptide | nuclear pore | FG domain | labile cross-β polymers

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Section: Methodsmentioning

confidence: 99%

Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Shi

Mori

Nizami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

222

191

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“…In our case, the STED laser is triggered by the pulsed supercontinuum laser operating at 38 MHz. STED beams at 775 nm wavelength are quite efficient for STED of fluorophores having peak emissions between 600 nm and 700 nm, a fact which can be used for multicolor recordings using a single-wavelength STED beam [14]. A notable advantage of a single STED beam approach is that the region in which the different fluorophores can assume the signaling state is defined by the very same doughnut.…”

Section: Methodsmentioning

confidence: 99%

A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

Görlitz¹,

Hoyer²,

Falk³

et al. 2014

PIER

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“…Recent advances open the possibility to resolve details at 20-50 nm and below (see examples in refs. [3][4][5][6][7]. In stimulated emission depletion (STED) nanoscopy (8,9)-as in all other fluorescence microscopy methods-the achievable resolution and contrast are largely determined by the total fluorescence signal, which is in turn limited by photobleaching.…”

mentioning

confidence: 99%

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Göttfert

Pleiner

Heine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.fluorescence nanoscopy | STED microscopy | photobleaching | superresolution

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Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

Cited by 273 publications

References 17 publications

Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

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Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

Cited by 273 publications

References 17 publications

Toxic PR n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Toxic PR n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

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Product

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Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export