Deuterated rhodamines for protein labelling in nanoscopy

Roßmann, Kilian; Akkaya, Kerem Can; Charbonnier, Corentin; Eichhorst, Jenny; Jones, Ben; Lehmann, Martin; Broichhagen, Johannes

doi:10.1101/2020.08.17.253880

Cited by 7 publications

(12 citation statements)

References 24 publications

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“…We next performed fluorescent labeling of BMP9 and TGFβ1, using a NHS-ester activated state-of-the-art silicon rhodamine derivate SiR-d12, which is characterized by high brightness and increased lifetime, suitable for confocal, life-time and super resolution microscopy (Roßmann et al, 2020). Both BMP9-SiR-d12 and TGFβ1-SiR-d12 bound their known high affinity receptor, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…In summary, we established an imaging platform that allows us to revise and test ligand receptor affinities in a cellular context. Due to the nature of the self-labeling Halo- and SNAP-tag, BMP and TGFβ receptors can now be visualized at a stoichiometric ratio using custom dyes, which meet the requirements for the respective experiments, e.g impermeability for surface-only stainings, increased photo-stability or STEDability (Birke et al, 2022; Roßmann et al, 2020). Future studies can now address (1) differences in ligand-receptor binding of monomeric receptors vs. receptor complexes, (2) binding behavior of other TGFβ-family ligands using the incorporation of unnatural amino acids at the C-terminus allowing site-directed coupling and positioning of fluorophores, (3) visualization of endogenous receptor populations in cells and in tissues using fluorescently labeled high affinity ligands as replacement for insufficient antibodies.…”

Section: Discussionmentioning

confidence: 99%

“…3. SiR-d12 labelled-BMP9 and -TGFβ1 allow for the identification of their respective high-affinity receptors To extend the studies to other members of the TGFβ ligand super-family, we labelled BMP9 and TGFβ1 using N-hydroxysuccinimide (NHS) silicon rhodamine-d12 (SiR-d12) esters (Nanda & Lorsch, 2014;Roßmann et al, 2020) (Fig. S4) to obtain far-red fluorescent BMP9-SiR-d12 and TGFβ-SiR-d12 (Fig.…”

Section: High-affinity Binding Of Activin a By Bmp Type II Receptorsmentioning

confidence: 99%

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A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

Jatzlau¹,

Burdzinski

Trumpp³

et al. 2022

Preprint

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The TGFβ superfamily of secreted growth factors comprises more than 30 members including TGFβs, BMPs and Activins. While all TGFβ superfamily members signal through heteromeric receptor complexes to regulate a plethora of developmental and homeostatic processes, each ligand possesses a unique affinity towards a subset of BMP and TGFβ type I and type II receptors. Whereas the Activin and TGFβ class display a higher affinity towards type II receptors, BMPs and GDFs preferentially bind to type I receptors. Sofar, the lack of specific antibodies and chemical biology tools hampered simultaneous testing of ligand binding towards all BMP and TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize the receptor complexes in dual color. In combination with novel fluorescently labeled TGFβ superfamily ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent growth factor binding for Activin A, TGFβ1 and BMP9 in a cellular context. We confirm ligand-receptor interface specificity by identifying BMPR2- or ALK2-mutants that switch from a low-affinity Activin A- or BMP9-receptor to a high-affinity receptor, respectively.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: High-affinity Binding Of Activin a By Bmp Type II Receptorsmentioning

confidence: 99%

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A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

Jatzlau¹,

Burdzinski

Trumpp³

et al. 2022

Preprint

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“…The need for custom-tailored dyes is in high demand as the range of microscopy modalities, experimental techniques and labelling strategies increases. Recent developments [9,[19][20][21][22] have focused mostly on boosting brightness, fluorescent lifetimes, chemical stability and/or fluorogenicity, the latter being a cause for cellular permeability. For the interrogation of cell surface proteins that are genetically fused to self-labelling protein tags (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Purified SNAP f and Halo was obtained as previously described. [20] Labelling dyes were dissolved in DMSO to a concentration of 1 mM and diluted in activity buffer (containing: 50 mM NaCl, 50 mM HEPES, pH=7.3 + 4 μg/mL BSA) to 500 nM. Protein was diluted in activity buffer to a concentration of 2 μM.…”

Section: Methodsmentioning

confidence: 99%

Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

Birke

Ast

Roosen

et al. 2021

Preprint

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Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.

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Chemical Biology Tools To Investigate Malaria Parasites

Broichhagen

Kilian

2021

ChemBioChem

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Parasitic diseases like malaria tropica have been shaping human evolution and history since the beginning of mankind. After infection, the response of the human host ranges from asymptomatic to severe and may culminate in death. Therefore, proper examination of the parasite's biology is pivotal to deciphering unique molecular, biochemical and cell biological processes, which in turn ensure the identification of treatment strategies, such as potent drug targets and vaccine candidates. However, implementing molecular biology methods for genetic manipulation proves to be difficult for many parasite model organisms. The development of fast and straightforward applicable alternatives, for instance small‐molecule probes from the field of chemical biology, is essential. In this review, we will recapitulate the highlights of previous molecular and chemical biology approaches that have already created insight and understanding of the malaria parasite Plasmodium falciparum. We discuss current developments from the field of chemical biology and explore how their application could advance research into this parasite in the future. We anticipate that the described approaches will help to close knowledge gaps in the biology of P. falciparum and we hope that researchers will be inspired to use these methods to gain knowledge – with the aim of ending this devastating disease.

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Deuterated rhodamines for protein labelling in nanoscopy

Cited by 7 publications

References 24 publications

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

Chemical Biology Tools To Investigate Malaria Parasites

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