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Pré-Publication, Document De Travail Année : 2021

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Charlotte Rimbault
Christelle Breillat
  • Fonction : Auteur
Benjamin Compans
Estelle Toulmé
  • Fonction : Auteur
Filipe Nunes Vicente
  • Fonction : Auteur
Monica Fernandez-Monreal
Patrice Mascalchi
  • Fonction : Auteur
Camille Genuer
  • Fonction : Auteur
Virginia Puente-Muñoz
Isabel Gauthereau
Eric Hosy
Gregory Giannone
Ingrid Chamma
Cameron Mackereth
Christel Poujol
Matthieu Sainlos

Résumé

ABSTRACT Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Dates et versions

hal-03402991 , version 1 (26-10-2021)

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Charlotte Rimbault, Christelle Breillat, Benjamin Compans, Estelle Toulmé, Filipe Nunes Vicente, et al.. Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes. 2021. ⟨hal-03402991⟩

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