Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques
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Evaluation Summary:
The precise, simple and non-interfering visualization of neuronal key structures is a major challenge and currently limiting the advancement of our understanding of brain function. This work presents intrabodies as selective and non-interfering tools for the visualization of PSD95 - a major scaffold of the neuronal excitatory postsynapse. This is an important and well executed work that provides an excellent new tool to study an important synaptic molecule.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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 recombinant 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.
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Evaluation Summary:
The precise, simple and non-interfering visualization of neuronal key structures is a major challenge and currently limiting the advancement of our understanding of brain function. This work presents intrabodies as selective and non-interfering tools for the visualization of PSD95 - a major scaffold of the neuronal excitatory postsynapse. This is an important and well executed work that provides an excellent new tool to study an important synaptic molecule.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is collaborative work from a number of groups with complementary expertise in the generation of binders, Biological Chemistry and Structural Biology and super resolution microscopy and Neuroscience. The groups have a long history of effective collaboration and are widely known to consistently produce high quality work.
The authors here build on previous (published) collaborative work in which they developed and characterised a number of small, non-antibody, protein binders to the important scaffold protein PSD-95, which through its PDZ-domains scaffolds AMPA-receptors via auxiliary proteins in the excitatory post synapse.
They now in the present work aim to establish these binders as tools to investigate PSD-95 function specifically and synaptic physiology globally by using the binders as fluorescent …
Reviewer #1 (Public Review):
This is collaborative work from a number of groups with complementary expertise in the generation of binders, Biological Chemistry and Structural Biology and super resolution microscopy and Neuroscience. The groups have a long history of effective collaboration and are widely known to consistently produce high quality work.
The authors here build on previous (published) collaborative work in which they developed and characterised a number of small, non-antibody, protein binders to the important scaffold protein PSD-95, which through its PDZ-domains scaffolds AMPA-receptors via auxiliary proteins in the excitatory post synapse.
They now in the present work aim to establish these binders as tools to investigate PSD-95 function specifically and synaptic physiology globally by using the binders as fluorescent markers for live and super resolution imaging and for the delivery of sensors for functional imaging. This is an important and worthy goal, as overexpression of fluorescent protein tagged PSD-95 is known to lead to artifacts and PSD-95 is an extremely important molecule to study.
The authors measure using NMR of in vitro reconstituted binding to purified components and a previously developed dimeric binder to the dual PDZ-domains of PSD-95 whether binding of the proposed binders interferes with binding of PSD-95 to stargazin, an auxiliary protein that mediates AMPA-receptor recruitment to synapses or whether engagement of the binding sites changes attachment of their binder. They find that neither is the case. They then move on to express their binders coupled to fluorophores under control of an expression system that prevents too strong overexpression and see that the binders become recruited to post synapses. They go on to test if the binders change synaptic physiology in measuring EPSCs and find no change. They then move on to test their binders in a number of super resolution modalities by linking them to fluorescent proteins and dyes. They furthermore use the binders to recruit a calcium sensor to the postsynapse, which they successfully use to detect and signalling in single synapses in dendritic spines.
The authors present very convincing data supporting their claims and provide very thorough characterization of the binders and their interaction with PSD-95 in live cells and during signalling events. The binders they present clearly do not interfere with PSD95 function as characterised and can be used to deliver dyes, probes, fluorescent proteins and sensors to functional postsynapses. This is an important step forward and very useful to the field.
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Reviewer #2 (Public Review):
The precise, simple and non-interfering visualization of neuronal key structures is a major challenge and currently limiting the advancement of our understanding of brain function. This work presents intrabodies as selective and non-interfering tools for the visualization of PSD95 - a major scaffold of the neuronal excitatory postsynapse. A toolbox of fully characterized modular imaging tools for use with various super-resolution microscopy techniques or to achieve synaptic enrichment of activity reporters of choice.
Here four key issues of current visualization approaches are addressed: The problem of visualizing exclusively the relevant protein target and not the closely related proteins. The problem that conventional probes, like antibodies, may introduce a significant labeling offset or display low …
Reviewer #2 (Public Review):
The precise, simple and non-interfering visualization of neuronal key structures is a major challenge and currently limiting the advancement of our understanding of brain function. This work presents intrabodies as selective and non-interfering tools for the visualization of PSD95 - a major scaffold of the neuronal excitatory postsynapse. A toolbox of fully characterized modular imaging tools for use with various super-resolution microscopy techniques or to achieve synaptic enrichment of activity reporters of choice.
Here four key issues of current visualization approaches are addressed: The problem of visualizing exclusively the relevant protein target and not the closely related proteins. The problem that conventional probes, like antibodies, may introduce a significant labeling offset or display low binding efficiency. The problem that live imaging approaches still largely rely on excessive genetic manipulation often including the artificial over-expression of a selected isoform or modification variant of the target protein. Finally, and possibly most importantly, the difficulty to visualize the target protein without interfering with its biological functions.
In contrast to chemical probes and fused fluorescent reporters the provided intrabodies are depending on promoters and regulated expression. As a result the probes are either over-expressed or they display a sub-stoichiometric target binding. As such this work also highlights the need for the development of improved systems to precisely control and adjust the expression level of such probes to avoid probe excess and, as a result of this, low contrast visualization of the target protein.
Due to the simplicity of application and the modular nature of the provided toolbox, it has the potential to be broadly used, especially in synergy with the emerging imaging techniques. Specifically, the verified target selectivity and the proven absence of functional interference can be expected to motivate the use of the provided PSD95 affinity proteins over the currently existing ones.
It is worth mentioning that the semi-rational development approach of the PSD-95 intrabodies seems to be applicable to other proteins of choice, enabling the production of new modular imaging tools.
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Reviewer #3 (Public Review):
Rimbault, Breillat, Compans et al., set out to characterize and exploit the use of previously described synthetic binders of PSD-95 as imaging tools for a wide range of techniques. They characterized the binding properties of these probes and tested the impact of these binders on PSD-95 function. The authors also evolved a set of these binders through addition of different fluorescent modules, as well as an expression regulation system to achieve close-to-endogenous labeling of PSD-95. The authors provide an extensive toolbox and an in-depth characterization of the tools used. The set of probes described in this manuscript will be of use to the neuroscience community, but some aspects of the specificity and application of the binders could be clarified.
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