Fluorogenic probes for live-cell imaging of the cytoskeleton

Lukinavičius, Gražvydas; Reymond, Luc; D’Este, Elisa; Masharina, Anastasiya; Göttfert, Fabian; Ta, Haisen; Güther, Angelika; Fournier, Mathias; Rizzo, Stefano; Waldmann, Herbert; Blaukopf, Claudia; Sommer, Christoph; Gerlich, Daniel W.; Arndt, Hans Dieter; Hell, Stefan W.; Johnsson, Kai

doi:10.1038/nmeth.2972

Cited by 778 publications

(859 citation statements)

References 28 publications

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“…The magnitude of the response parallels the increase in fluorescence intensity of the tagged dyes 1 a – k ‐Halo, bound nonspecifically and reversibly to bovine serum albumin, upon addition of the anionic surfactant sodium dodecyl sulfate (SDS, see Figure S4), as shown previously for SiR 6h,6k. Interestingly, when the cationic surfactant cetyltrimethylammonium bromide was added instead of SDS, the fluorescence intensity decreased (Figure S4), suggesting that the fluorogenic response of the dyes 1 g , 1 h , and SiR may (at least partially) be due to interactions of the positively charged tricyclic fluorophore core with the local negative charges on the HaloTag protein surface in immediate proximity to its active site.…”

supporting

confidence: 74%

“…Whereas SiR is excited at λ ≈640 nm, and its fluorescence is detected at 650–700 nm, 580R and 580CP can be excited at λ ≈560 nm or 590 nm and detected at 605–625 nm, enabling clear color separation for 580R (Figure S5) with low crosstalk between the channels. Figures 4 and S17 show two‐color STED images of living HeLa cells (vimentin fibers labeled with 580R or 580 CP , respectively; tubulin labeled with a SiR tubulin probe) 6k. As evident from both Figures, a larger cross section for stimulated emission of the red‐shifted dye at the STED wavelength results in higher optical resolution for the long‐wavelength ( SiR ) channel.…”

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confidence: 96%

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Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

et al. 2016

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A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm.

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confidence: 74%

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confidence: 96%

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

et al. 2016

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“…4C′). To test whether this aggregation was an artifact of the labeling system, we stained oocytes using a silicon-rhodamine-conjugated microtubule probe (SiR-tubulin) (38). We found no aggregates in either control or Khc mutA oocytes using SiR-tubulin (Fig.…”

Section: Sliding Between Cortically Anchored Microtubules and Free Cymentioning

confidence: 95%

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

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Lakonishok

et al. 2016

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

122

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Cytoplasmic streaming in Drosophila oocytes is a microtubulebased bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.kinesin-1 | microtubules | cytoplasmic streaming | Drosophila | axis determination

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“…These fluorescent dyes may be easily conjugated with a marker protein or protein of interest, and since their emission spectra lies in near-infrared, they are excellent for super-resolution microscopy techniques. Later, SiR-actin and SiR-tubulin were generated and demonstrated to be effective in animal fibroblasts (Lukinavičius et al, 2014). However, no attempt has yet been made to apply these labels to plant systems.…”

Section: Plant Sciencementioning

confidence: 99%

Visualization and analysis of actin cytoskeleton organization in plants

Pozhvanov

2018

BioComm

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The plant cytoskeleton is a highly dynamic system that consists of two components: microfilaments and microtubules. Actin microfilaments are essential for polar growth, cytoplasmic streaming, directing polar growth, anchoring the nucleus, gravity sensing, signalling pathway integration and a number of other functions. Actin morphology and dynamics are orchestrated by a variety of small actin binding proteins, and some of them have become a source of actin interaction domains widely used as markers for microfilaments in fusions with fluorescent reporter proteins. However, older techniques are still employed for actin visualization. In this short review, we will focus on the diversity of fluorescent reporter fusions for F-actin and on approaches and existing free software for the analysis of cytoskeleton organization, mainly in Arabidopsis.

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Fluorogenic probes for live-cell imaging of the cytoskeleton

Cited by 778 publications

References 28 publications

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Visualization and analysis of actin cytoskeleton organization in plants

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