Noncanonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

Reviewer #1 (Public review):

Summary:

The authors utilize genetic code expansion to tag TDP-43 and G3BP1, and evaluate this protein tagging system (ANAP) compared to antibodies, and evaluate protein trafficking and stress granule formation in response to stress with sodium arsenite treatment. They find similar staining to antibodies in HeLa cells, mouse embryonic stem cells, and primary mouse cortical neurons. This is a useful study that demonstrates the utility of ANAP tagging to evaluate ALS proteins.

Strengths:

Rescue of cell survival by ANAP-tagged TDP-43 is compelling

Weaknesses:

While the ANAP-tagged proteins had similar distributions to antibody staining, there were some discrepancies that may be more explained by the technique than by novel findings, as the authors suggested. The inclusion of additional controls to evaluate this would be helpful.

Reviewer #2 (Public review):

Summary:

In this manuscript, Chen and colleagues describe a novel means of labeling two RNA-binding proteins, G3BP1 and TDP-43, using genetic code expansion. Overexpressed constructs that incorporate the intrinsically-fluorescent non-canonical amino acid Anap redistribute to cytoplasmic granules upon application of external stressors such as sodium arsenite. Similar labeling and redistribution of overexpressed G3BP1 and TDP-43 were observed in cultures of mouse primary neurons.

Strengths:

Genetic code expansion and non-canonical amino acid labeling have quite a few advantages over traditional fusion proteins for tracking protein redistribution in living cells. The authors show that they are able to label exogenous G3BP1 and TDP-43 with the non-canonical amino acid Anap and follow labeled proteins in living cells with and without stress.

Weaknesses:

The authors do not convincingly leverage the advantages of genetic code expansion in the current study. There is no specific question posed by the authors that can be or is answered using this approach, and several of the experiments lack critical controls. This is also not the first example of TDP-43 labeling by genetic code expansion (see PMID: 38290242). As a result, the study as a whole adds little to our understanding of protein trafficking and behavior under stress.