PREreview of Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

Dear authors,

thank you for sharing this preprint with everyone. I recently decided to support research in chemical biology by providing feedback and thoughts on preprints in this field. Your preprint caught my eye, and I hope you will find the comments below useful (I hesitate to call this peer review, but if you would like to share what I wrote with a journal please feel free).

Kind regards,

Milka

Comments to authors:

In this preprint the authors combine genetic code expansion (a strategy that allows one to incorporate non-canonical amino acids at a specific position within the protein), with biorthogonal labeling (a type of chemical reaction executed in living systems in a way that is orthogonal to biological/physiological reactions) and optical microscopy to visualize two members of the transmembrane AMPA receptor (AMPAR) regulatory protein (TARP) family, 2 and 8 in live neurons and brain slices.

The main problem that the authors set out to address is as follows:

- The authors present evidence that antibodies can’t recognize endogenous TARP gamma2 and gamma8 in neurons because these proteins are found to be associated with AMPAR’s ligand binding domain (LBD) in a manner that masks the epitope. This suggests that strategies like immunostaining using fluorescently labeled antibodies are not appropriate for TARP imaging. Therefore, to solve this problem the authors decided to pursue a completely different strategy that does not rely on antibody use. Overall, this is an important research problem and the approach the authors chose to pursue is appropriate for addressing this problem.

Summary of the approach:

- The authors incorporated clickable trans-cyclooctene derivatized lysine into TARP gamma2 and gamma8 using established strategy for genetic code expansion. Exposing the modified TARP gamma2 and gamma8 to cell-impermeable tetrazine-dyes resulted in biorthogonal reaction called strain-promoted inverse electron-demand Diels-Alder cycloaddition reaction (SPIEDAC), whereby TARP gamma2 (or gamma8) featuring a modified lysine is covalently labeled with the fluorescent dye. The strategy does not seem to prevent TARP gamma2 and gamma8 from interacting with AMPARs, or affect TARP gamma2 and gamma8 function making this a well-tolerated modification. Additionally, the fluorescent signal is strong enough to allow visualization in primary neurons, as well as organotypic hippocampal slice cultures.

Summary of key biological observations:

- The key observations made in this study are:

o Most of endogenous TARP gamma2 and gamma8 are found to be associated with AMPAR at the surface of hippocampal neurons.

o TARP gamma2 was found to be mostly localized to synaptic spines as opposed to extrasynaptic sites, while TARP gamma8 had a more even distribution between the two, while still favoring synaptic spines.

o Analysis of visual data allowed for identification of TARP gamma2 synaptic clusters (80 nm in size) that localized within dendritic spines. TARP gamma8 was not observed to form clusters under the conditions used in this work.

Concluding remarks:

Overall, I found the application of the genetic code expansion in this neurobiology context to be of interest and I can see the potential of this methodology (with further improvements elaborated on by the authors in their Discussion section) for imaging systems that are otherwise difficult to address using optical microscopy.