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

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.

High-Resolution Fluorescence Imaging Combined With Computer Simulations to Quantitate Surface Dynamics and Nanoscale Organization of Neuroligin-1 at Synapses

“…Biotinylated AP-NLGN1 was densely labeled with Alexa647-conjugated mSA in live conditions, followed by fixation, and the stochastic emission of single fluorophores was induced ( Figures 4C,E ). When super-resolved images were reconstructed from individual detections, NLGN1 filled PSDs labeled with the Xph20-GFP intrabody to PSD-95 ( Rimbault et al, 2019 , 2021 ) without forming any specific sub-domain ( Figures 4G,I ), as previously reported ( Chamma et al, 2016a ). In the dendritic shaft, NLGN1 showed a fairly homogeneous membrane localization, likely corresponding to the fast-diffusing molecules detected live by uPAINT.…”

“…Control unbleached synapses did not display any significant drop in NLGN1-GFP fluorescence, revealing negligible observational photobleaching. Additional FRAP experiments with a lower sampling rate in neurons co-expressing NLGN1-GFP and Xph20-mRuby2, an intrabody specific to PSD-95 ( Rimbault et al, 2019 , 2021 ), showed that photobleached NLGN1-GFP was essentially post-synaptic ( Supplementary Figure 3A ). Furthermore, the NLGN1-GFP fluorescence recovery after 1 h was 60%, a value in line with the first round of experiments performed at higher sampling rate ( Supplementary Figures 3B,C ).…”

“…shRNA-resistant AP-NLGN1 was described earlier ( Chamma et al, 2016a ; Toledo et al, 2022 ). The specific intrabody to PSD-95, Xph20 (Addgene ID 135530) was described recently ( Rimbault et al, 2019 , 2021 ), and we used both GFP- and mRuby-tagged versions. The bacterial production of mSA, purification, and conjugation to organic dyes (STAR635P or Alexa647) to a final DOL comprised between 0.6 and 2 (dye to protein ratio), were described previously ( Chamma et al, 2017 ).…”

Distinct in vivo dynamics of excitatory synapses onto cortical pyramidal neurons and parvalbumin-positive interneurons

“…The ENABLED strategy provides a viable way to visualize endogenous proteins that is potentially expandable to other proteins. There are also parallel efforts to develop alternative strategies to label endogenous proteins for live imaging ( Gross et al, 2013 ; Mikuni et al, 2016 ; Nishiyama et al, 2017 ; Rimbault et al,2019 , 2021 ; Suzuki et al, 2016 ; Zhong et al, 2021 ). Although each of these strategies has its own strengths and limitations, endogenous protein imaging will likely constitute an important research avenue in the future.…”

Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation

“…Two approaches were applied for the fluorescence imaging of proteins. We transfected primary neurons with a plasmid coding for Xph20-eGFP, a fluorescent molecular construct designed to bind the postsynaptic scaffold protein PSD-95 (Rimbault et al, 2021). The second method is the direct labeling of tubulin and/or F-actin with silicon-rhodamine (SiR) dyes designed for STED microscopy (Lukinavičius, 2016).…”