Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage

Round 1

Reviewer 1 Report (Previous Reviewer 1)

I have just one comment for point #6.

I am aware that immunofluorescence and PLA are different, but both detect primary antibodies. Therefore, as the induced Ku70-HA immunofluorescence (3C, detecting all Ku-70 in cell) shows diffuse staining, it begs the question of what the discrete foci that Ku-80 is binding to are biologically? So Ku-80 must be bound to a sub-population (very small fraction) of Ku-70 in 3D which is what is detected and amplified by PLA?

I understand that those are places that contain a heterodimer of 70/80 and that this is a positive control but then why do we not see foci when you just look at 70? HA antibody should detect both the foci and the diffuse staining unless the authors think it is too weak and not amplified enough?  Since 70/80 are known to bind DSBs or ends of DNA, are those breaks? The only difference between indirect immunofluorescence and PLA is detecting one antibody vs. two that are in close proximity. So what is biologically represented by the ku70/80 foci? The authors' response of "we expect 3C to be different than 3D" is unclear to the reader for biologically addressing what the foci is. Especially, if they exist as a heterodimer, you would expect to see Ku-70/80 localization to look similar. For e.g. a telomere protein, when over expressed, will only go to the telomere and be present as a foci albeit with higher background, whether you look at it as indirect immunofluorescence or PLA in conjugation with a binding partner- both look similar.

This could be a trivial explanation but still necessary since this serves as the positive control for all other experiments. A few sentences should suffice.

 

Author Response

Immunofluorescence, such as the one shown in Figure 3C, allows identification of all HA-Ku70 proteins that are bound by the HA antibody. So a protein like Ku70 which is present all through the nucleus, will be visualized as a diffuse signal since it is present everywhere in the nucleus and the resolution is not sensitive enough to detect individual proteins.

PLA detects 2 proteins that are in close proximity, but only when the 2 proteins have their respective antibodies bound to each of them. And then, the 2 antibodies have to be recognized by 2 secondary antibodies, which have DNA oligos attached, and then these oligos need to be amplified successfully to then be recognized by complementary oligos coupled to fluorochromes, yielding a signal visualized in the form of a strong dot. The signal that results from this is much stronger than the signal from a single antibody recognizing 1 protein such as in the case of the indirect immunofluorescence, because the PLA signal is amplified by the rolling-circle amplification (RCA), which allows 1000-fold amplification (hence visualized as a strong dot, or focus). However, because 3 different steps have to be successfully completed before obtaining a signal, it is not as successful as a general hybridization with 1 antibody, thus the number of dots does not accurately reflect the actual the number of proximal protein pairs in the cell.

Reviewer 2 Report (Previous Reviewer 2)

Authors took the comments seriously and revisited most of the criticised comments which improved the manuscript significantly. one minor point to be considered. it would be nice if authors quantitate the PLA foci and show it in graph. 

Author Response

We thank the reviewer for their positive comments on our work. We quantified the PLA foci shown in 3 different experiments, and the graphs were included in the revised version of the manuscript as follows: the PLA foci shown in Figure 3C were quantified/graphed in Supplemental Figure S3B. The PLA experiment shown in Figure 4A was quantified in the graph in Figure 4B. And the PLA foci shown in Figure 5B are quantified are shown in the graph in Figure 5C. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This paper uses proximity-dependent biotin identification (BioID2) to identify phospho-specific interacting proteins of Ku70, a member of the DNA damage response pathway. Utilising this technique is a unique way of identifying such specific interactions and as such represents a novel direction in this paper. However, the supporting evidence need further clarification before full confidence can be afforded to the findings. There are several areas where the paper requires the following: more consistent quantification of the data, comparison of the various BioID2 datasets given this is the first report of such a technique (supplemental tables were somehow missing from the materials provided), and most importantly the representative images evidencing such specific interactions need to be provided in a way that can be evaluated by readers. As of now, crucial images are provided at too low magnification to be able to corroborate any of the quantifications in the graphs. Thus, although the paper is promising,  not all of the data can be evaluated to support the authors claims in its current form. If the authors provide the proper images, quantifications, data set comparisons, and interpretation of data, then the paper can be considered for publication. Some critical points that absolutely should be addressed are below.

 

Major comments:

 

1.    Figure 1B: The authors say that 8 hours is a good time point to add biotin since the protein is expressed by then. They only show a time course for the Ku70-S155A-BioID2 cell line but mention both (line 107). It makes more sense to show both at the same time, especially since the main cell line in question is S155D and phosphomimetic may affect expression.

 

2.    Critical point: For all figures showing immunofluorescence/PLA, insets (zoomed in images) of single cells with the HA/PLA channel in white should be shown. The authors should include single channel pictures as white colored, instead of green/red to be inclusive of all scientist readers. As of now the figures are too small to assess signal properly for the reader, so proper high magnification images should be provided. This should absolutely be done for the following figures since the foci are so small that nothing can be evaluated without single cell insets:

Figure 1C, Figure 3C, D, Figure 4A and Figure 5A, B. 

 

3.    Especially for Figure 1C, S155A and D cell morphology looks abnormal, which could be expected for S155D but is surprising for S155A with increased survival time. Is this expected or just a fluke from selected images? 

 

4.    It is unclear how much overlap is there between the background Ku70-WT endogenous control and the S155A and D cell lines for BioID protein targets. Should we expect to see some phospho specific targets in that subgroup? Also, we would expect more overlap between the Ku70 S155A and Ku70 WT group perhaps. Those Venn diagrams would be helpful to include to assess the accuracy of BioID for this purpose. I did not see Tables S1-3 provided, only the Figures S1 and S2 (Line 553), which is unfortunate and may be a glitch but it should be remedied.

 

5.    Strikingly, the subtraction of the negative controls from S155A, leaves only 3 targets with 2 shared between this approach and the SAINT scores. But surprisingly, those two do not have the highest SAINT scores, which is mysterious. Can the authors comment on this, especially as the choice of TRIP12 is made from the high SAINT score so it begs the question of how accurate it is? Why not just perform SAINT analysis on the subtracted protein set since the two are intersected at the end anyway? 

 

6.    Figure 3C and D: The two cell lines in 3C appear to be expressing protein that is just present all over the nucleus. I cannot make out any foci, just uniform staining, which is perhaps expected without DSBs. However, as I understand the PLA in 3D shows foci of Ku80 localising to Ku70? What are these foci then? Why does the PLA look different than the induced HA? Also, the foci in the Ku80 control should be counted and reported as was done for Figure 4 and Supp Figure S1 to be consistent (# of foci and % of cells with foci).

 

7.    Figure 4A: I do not see any foci at all in the Cy3 channel which is in red. Single channels should be shown in white. As of now, I do not see the single, or 4 foci differences the authors claim in the text or quantify in the graph as being represented in the images. Without these images presenting a strong case, it is hard to believe the author’s claims of TRIP 12 in vivo interactions, given the high 0.99 SAINT score.

 

8.    Ku70 -HA has 1.60 foci in induced case in the 4B graph. How does this compare to the Ku80 positive control numbers?

 

9.    Figure 5A: Should we expect foci for H2A.X? It looks uniform for the 10 gy, which may be too high to see foci. Yet again, it would be easier to see single channels in white with insets, given this is very important to support the author’s claims.

 

10. Again, in 5C, the readers are left to believe the graph for increase in number of TRIP12 foci (to 15) since the images are too zoomed out to see any foci. Claims cannot be substantiated without explicit representative image support.

 

11. Is there an antibody to CHD3? Could the authors check that one as well? It would strengthen the appeal of this technique as a method for phospho-specific interaction screening and elevate the paper considerably. Another possible positive control could be PLA with the Aurora B/Chromosomal Passenger Complex antibodies. Is it surprising to the authors that Aurora B is not in the 17 proteins interacting with S155D? Should it not be there? 

 

 

Reviewer 2 Report

In this manuscript Abbasi et al, identified TRIP12 as Ku70 interacting partner using BioID approach. Authors also claim that TRIP12 specifically interacting with phosphomimic mutant of Ku70 (Ku70 S155D).  Though the TRIP12 interaction with Ku70 after DNA damage seem interesting, authors failed to show biological significance of TRIP12 and Ku70 interaction. more experiments need to be done to consider this manuscript for publication

1. Authors should show TRIP12 and Ku70 interaction by immunoprecipitation using both phospho mutants in presence and absence of DNA damage. 

2. does this mutations affect Ku70 binding to other NHEJ components? and recruitment to DNA damage site?

3. Figure 4A, authors should do PLA using phospho dead Ku70 S155A as an internal control.

4. Figure 4B, authors should counterstain the PLA using gH2AX ab (raised in either goat or sheep) and show PLA foci colocalization with gH2AX. this confirms TRIP12 and Ku70 interaction is specific at DNA damage site and not  random.

5. what is the functional significance of TRIP12 and Ku70 interaction?