Peer review of: Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy (2019)

RR2
 REVIEWERS' COMMENTS:
Reviewer #1 (Remarks to the Author):
Reviewer #2 (Remarks to the Author):
Attached below:
  The changes and additions made to the manuscript in this revision have clarified many points and
 strengthened the evidence in places where it was insufficient, and all of the points I raised have
 been answered to my satisfaction. —PC
  The authors have sufficiently addressed my concerns. I have attached one comment regarding an
 response that could be addressed in the text.
 
The authors state that “post-expansion labeling protocols substantially increases the epitope accessibility for IgG antibodies resulting in higher labeling densities especially of sterically demanding multiprotein complexes” This is a significant claim and perhaps one of the most important takeaways from the paper, as super-resolution imaging is fundamentally limited by labeling density. Can the authors provide quantitation to this point? Specifically, does ExM-SIM offer higher labeling densities than STORM in 1 color (STORM is prelabeled). If so, can higher resolutions be achieved, or are there more continuous structures. Can a labeling density metric be derived?
We fully agree with the reviewer that our finding of the advantage of post-expansion labeling regarding labeling efficiency especially of sterically demanding multiprotein complexes is one of the most important results of our manuscript. The comparison of labeling density of unexpanded versus expanded samples is, however, a very complex endeavor. First, the antibody binding constant can differ after protein denaturation as used by MAP. Hence, the binding efficiency of the same antibody can vary before and after expansion. Second, SIM is per se not single-molecule sensitive as (d)STORM is thus rendering the direct comparison difficult. In fact, only the labeling continuity and signal density along a filamentous structure like the SC can be compared, and this is what we did to compare the different expansion protocols. Due to this reasons the advantage of post-expansion labeling might be less noticeable in most experiments. Only in sterically demanding very protein dense regions of multiprotein complexes the epitope accessibility might be improved upon expansion, presupposed that the epitopes survive denaturation and expansion and are still recognized by the antibody. In the case of the SC the multilayered organization of proteins in the central element and SYCP3 fraying of the axes represent such cases where post-expansion labeling outperforms in some regions pre-expansion labeling. To cut a long story short, we fully agree with the reviewer and acknowledge his/her ideas to further increase the impact of our manuscript but the question is presently too complex to be answered correctly. The other question whether post-expansion labeling can achieve a higher resolution has to be answered with yes mainly due to the fact that the linkage error (the distance between the fluorophore and the protein of interest) decreases substantially when using post-expansion labeling. If as in our studies, proteins are labeled by immunolabeling pre-expansion the linkage error is ~ 20-30 nm (primary and secondary IgG antibody with a size of 10-15 nm). This linkage error will “expand” during expansion – in our case ~ 4 fold to 80-120 nm whereas post-expansion labeling will reduce the ‘true’ linkage error to ~ 5-8 nm for a 4-fold expanded sample. We highlighted these findings in more detail in the revised version of our manuscript.
I accept the authors claim that this may be beyond the scope of this manuscript. However, could the authors elaborate on this in the discussion here:

“Our results demonstrate that post-expansion labeling protocols reduce the linkage error and substantially increase the epitope accessibility for IgG antibodies resulting in higher labeling densities especially of sterically demanding multiprotein complexes14,29”
It's probably difficult to claim substantially increased epitope accessibility without actually making the comparison. I think a less strong claim (i.e. more continuous labeling suggests higher accessibilities, although this is a challenging expt and requires future measurements in different epitope contexts).

 Reviewer #3 (Remarks to the Author):
 The revised version of the manuscript carefully addresses the issues that I raised previously.
 Especially, the new fitting schemes for the distance histograms for quantifying the strand distance
 is well validated with simulation of a predefined helical structure and resulted in consistent results
 for both 1- and 2-color images. Also, the pre- and post-expansion alignment is properly executed
 and highly informative.
 However, I found a few points in the revised manuscript that have to be corrected or addressed.
 (1) The following paragraph added to the revised manuscript contain inconsistent figure call-outs.
 “Fig. 4d” should be corrected to “Fig. 4g”. “Fig. 4g and Supplementary Fig. 10” should be corrected
 to “Fig. 4d”.
 In some lateral view sections of MAP-SIM images of the SYCP3 signal the N-terminus of the
 transverse filament protein SYCP1 and the central element protein SYCE3 revealed bi- and
 multimodal signal distributions supporting a multilayered organization of the central element (Fig.
 4d and Supplementary Fig. 10). On the other hand, averaged cross-sectional intensity profiles of
 SYCE3 and SYCP1N (Figs. 4e,f) clearly showed a monomodal distribution of the two proteins (Fig.
 4g and Supplementary Fig. 10)in accordance with the unstructured organization of the transverse
 filaments into a large single layer as revealed by EM tomography in a previous study27.
 (2) The author’s reply to my major comment #5 is largely satisfactory. However, I cannot find the
 “new paragraph” that the authors mentioned in the last sentence of their reply. I just found one
 new sentence in the revised paragraph. Where is the “new paragraph”? Since the journal does not
 limit the length of the texts, can the authors consider adding some of the rebuttal comments in the
 manuscrip to help readers infamiliar to the biological system like myself?
 (3) The author’s comment to my minor comment (c) about DNA-PAINT’s requirement for TIRF is
 correct. However, TIRF is compatible with their particular application of imaging SCs spread out on
 the coverslip. Therefore, for many systems to which TIRF is applicable, including SCs, I don’t see
 the advantage of MAP-SIM over Exchange-PAINT.
 In addition, MAP-SIM depth is shorter than regular SIM. Theoretically, MAP-SIM depth is the SIM
 depth divided by the expansion factor. For instance, the working distance of a high-NA objective
 for SIM is ~200 micron. Regarding an expansion factor of 4, the depth limit would be ~50 micron.
 Moreover, the spherical abberation of the expanded gel may be different from unexpanded
 samples in aqueous media. It is true that even after considering expansion and abberation, the
 imaging depth of MAP-SIM is better than DNA-PAINT. It is noteworthy to mention these for guiding
 readers considering MAP-SIM for their systems of interest.