A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

Vladimir Khayenko; Clemens Schulte; Sara L. Reis; Orly Avraham; Cataldo Schietroma; Rafael Worschech; Noah F. Nordblom; Sonja Kachler; Carmen Villmann; Katrin G. Heinze; Andreas Schlosser; Ora Schueler-Furman; Philip Tovote; Christian G. Specht; Hans M. Maric

doi:10.1002/anie.202202078

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

Vladimir Khayenko, Clemens Schulte, Sara L. Reis, Orly Avraham, Cataldo Schietroma, Rafael Worschech, Noah F. Nordblom, Sonja Kachler, Carmen Villmann, Katrin G. Heinze, Andreas Schlosser, Ora Schueler-Furman, Philip Tovote, Christian G. Specht^*, Hans M. Maric^*

^*Corresponding author for this work

Department of Microbiology and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

Original language	American English
Article number	e202202078
Journal	Angewandte Chemie - International Edition
Volume	61
Issue number	30
DOIs	https://doi.org/10.1002/anie.202202078
State	Published - Jul 2022

Bibliographical note

Funding Information:
We thank Dr. Jens Vanselow, Stephanie Lamer and Alvaro Ciudad for supporting the mass spectrometric studies and analysis. We thank Prof. Eric Allemand and Dr. Fabrice Ango for providing gephyrin isoform constructs. And we thank Dr. Katharina Hemmen, Dr. Hanna Heil, Mike Friedrich and Jürgen Pinnecker for their guidance in 3D image analysis and light microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (DFG MA6957/1‐1) to H.M.M.; Agence Nationale de la Recherche (InVivoNanoSpin, ANR‐20‐CE11‐0002‐02) to C.G.S.; Deutsche Forschungsgemeinschaft (Project ID 424778381 – TRR 295), Heisenberg professorship and project funds to P.T. (TO 1124); PhD fellowship from Fundação para a Ciência e a Tecnologia to S.L.R. Open Access funding enabled and organized by Projekt DEAL.

Publisher Copyright:
© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

Keywords

Dimerization
Fluorescent Probes
Neuroscience
Peptides
Super-Resolution Microscopy

Access to Document

10.1002/anie.202202078

Cite this

Khayenko, V., Schulte, C., Reis, S. L., Avraham, O., Schietroma, C., Worschech, R., Nordblom, N. F., Kachler, S., Villmann, C., Heinze, K. G., Schlosser, A., Schueler-Furman, O., Tovote, P., Specht, C. G., & Maric, H. M. (2022). A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**. Angewandte Chemie - International Edition, 61(30), Article e202202078. https://doi.org/10.1002/anie.202202078

@article{277a66e1007545098550905aa4eda49a,

title = "A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**",

abstract = "Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.",

keywords = "Dimerization, Fluorescent Probes, Neuroscience, Peptides, Super-Resolution Microscopy",

author = "Vladimir Khayenko and Clemens Schulte and Reis, {Sara L.} and Orly Avraham and Cataldo Schietroma and Rafael Worschech and Nordblom, {Noah F.} and Sonja Kachler and Carmen Villmann and Heinze, {Katrin G.} and Andreas Schlosser and Ora Schueler-Furman and Philip Tovote and Specht, {Christian G.} and Maric, {Hans M.}",

note = "Funding Information: We thank Dr. Jens Vanselow, Stephanie Lamer and Alvaro Ciudad for supporting the mass spectrometric studies and analysis. We thank Prof. Eric Allemand and Dr. Fabrice Ango for providing gephyrin isoform constructs. And we thank Dr. Katharina Hemmen, Dr. Hanna Heil, Mike Friedrich and J{\"u}rgen Pinnecker for their guidance in 3D image analysis and light microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (DFG MA6957/1‐1) to H.M.M.; Agence Nationale de la Recherche (InVivoNanoSpin, ANR‐20‐CE11‐0002‐02) to C.G.S.; Deutsche Forschungsgemeinschaft (Project ID 424778381 – TRR 295), Heisenberg professorship and project funds to P.T. (TO 1124); PhD fellowship from Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia to S.L.R. Open Access funding enabled and organized by Projekt DEAL. Publisher Copyright: {\textcopyright} 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.",

year = "2022",

month = jul,

doi = "10.1002/anie.202202078",

language = "American English",

volume = "61",

journal = "Angewandte Chemie - International Edition",

issn = "1433-7851",

publisher = "John Wiley and Sons Ltd",

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Khayenko, V, Schulte, C, Reis, SL, Avraham, O, Schietroma, C, Worschech, R, Nordblom, NF, Kachler, S, Villmann, C, Heinze, KG, Schlosser, A, Schueler-Furman, O, Tovote, P, Specht, CG & Maric, HM 2022, 'A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**', Angewandte Chemie - International Edition, vol. 61, no. 30, e202202078. https://doi.org/10.1002/anie.202202078

TY - JOUR

T1 - A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

AU - Khayenko, Vladimir

AU - Schulte, Clemens

AU - Reis, Sara L.

AU - Avraham, Orly

AU - Schietroma, Cataldo

AU - Worschech, Rafael

AU - Nordblom, Noah F.

AU - Kachler, Sonja

AU - Villmann, Carmen

AU - Heinze, Katrin G.

AU - Schlosser, Andreas

AU - Schueler-Furman, Ora

AU - Tovote, Philip

AU - Specht, Christian G.

AU - Maric, Hans M.

N1 - Funding Information: We thank Dr. Jens Vanselow, Stephanie Lamer and Alvaro Ciudad for supporting the mass spectrometric studies and analysis. We thank Prof. Eric Allemand and Dr. Fabrice Ango for providing gephyrin isoform constructs. And we thank Dr. Katharina Hemmen, Dr. Hanna Heil, Mike Friedrich and Jürgen Pinnecker for their guidance in 3D image analysis and light microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (DFG MA6957/1‐1) to H.M.M.; Agence Nationale de la Recherche (InVivoNanoSpin, ANR‐20‐CE11‐0002‐02) to C.G.S.; Deutsche Forschungsgemeinschaft (Project ID 424778381 – TRR 295), Heisenberg professorship and project funds to P.T. (TO 1124); PhD fellowship from Fundação para a Ciência e a Tecnologia to S.L.R. Open Access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

PY - 2022/7

Y1 - 2022/7

N2 - Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

AB - Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

KW - Dimerization

KW - Fluorescent Probes

KW - Neuroscience

KW - Peptides

KW - Super-Resolution Microscopy

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U2 - 10.1002/anie.202202078

DO - 10.1002/anie.202202078

M3 - Article

C2 - 35421279

AN - SCOPUS:85129447327

SN - 1433-7851

VL - 61

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

IS - 30

M1 - e202202078

ER -

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

Abstract

Bibliographical note

Keywords

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