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

Peer Review File 

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
This manuscript presents a comparison between different high-resolution microscopy modalities 
involving sample expansion, using the well-characterized dimensions of the synaptonemal complex 
as a standard. Through innovations in many steps of sample and gel preparation, the authors 
demonstrate that expansion microscopy followed by 3D structured illumination results in optimal 
sample preservation, resolution equivalent to the best non-expanded single-molecule localization-
based microscopy, and the ability to image 3 (or more) color channels. The synaptonemal complex 
images are truly stunning, and compare with some of the best light and electron microscopy 
images yet taken. 
 
Of special note are the clear recapitulation of axis fraying (resolved well by MAP-SIM and shown to 
be SYCP3-positive) and occasional doubling observed of single lateral elements. The ability to 
observe these rare and very small-scale structural features with immunofluorescence is very 
exciting, and demonstrates that methods such as MAP-SIM are poised to open some new areas of 
investigation in cell biology. 
 
While the images speak for themselves to the high quality of the methods, the structure of the 
manuscript itself is often not as clear. Figures 1–3 are straightforward and mostly solid 
recapitulations of previous work, with the exception of the novel light-microscopy visualization of 
split signals of a single SYCP3 axis in 2f. _It would be very informative if we were told how often 
and under what circumstances this phenomenon can be visualized._ 
 
For the other figures, it is unfortunately often difficult to assess the connection between the figure 
and the text. The main recurring problem is that figure call-outs in the main text tend to lack 
descriptions and subpanel labels (i.e., not just "Figure 4" but "Figure 4b" is better when 
appropriate) to aid the reader. Figure 4, for example, is only referred to once, in a rather general 
way, in the main text, although it has 7 subpanels that make different points between them. 
Readers are left to Figure it out for themselves, which makes the paper somewhat challenging to 
read in parts. 
 
Major comments: 
 
The whole section from lines 260-286 is rather difficult to understand as it is written; further, it 
makes the claim "MAP-SIM discloses new information about the molecular organization of the SC" 
but does not say clearly what this new information is. Stating that your method "indicated a far 
more complex distribution of the central region proteins" does not tell me what is new. This section 
should be rewritten with a focus on concretely describing what has been found out, to the extent 
this is possible. 
 
Within this section, in lines 273–274: “the signal distributions of the MAP-SIM imaged SYCP1N-
terminus and SYCE3 indicated a far more complex distribution of the central region proteins than 
the bimodal distribution previously described by dSTORM and immunogold EM” -- although no 
figure panel is mentioned, I assume this is referring to the image in Figure 4c (unfortunately 
obscured by the unnecessary red SYCP3 signals) and the corresponding bimodal pattern of profile 
#2 in Figure 4d. However, given the small sample size here (one profile), the novelty of the 
method, and the fact that the averaged profiles show a clearly monomodal distribution of SYCP1N 
and SYCE3, is it possible that this bimodal profile is an artifact/noise? It may not be, but some 
evidence should be provided or at least the possibilities discussed. 
 
In Fig. 4e’s legend: “Frontal views of the two proteins used for cross-sectional profiles are shown 
in yellow” - it is not clear what this means; does this mean that yellow is a false-color overlay on 
the regions selected for LineProfiler analysis? I suggest using arrows or brackets to show these 
regions, instead of a false color, which prevents a clear view of the image itself (for instance, using 
magenta/green alone would allow an appreciation of the SYCE1/SYCP1N signals in the absence of 
the red SYCP3 signals from a,b, and c). Even better would be to point out these regions in 4a so 
we can see where they lie with respect to the twisting of the lateral elements. 
 
Since the Lineprofiler analysis seem to be 2d only, some discussion should be included in the text 
of how this could tend to underestimate the true peak-to-peak separation, since helical twisting of 
lateral elements will bring their XY projections closer together than their true distance. Some part 
of this discussion is found in the legends for supplemental Figure 2, but perhaps it is an important 
enough point to be mentioned in the main text. 
 
 
Minor comments: 
 
For the LineProfiler analysis, please clarify at what point the input image is reduced from 3D to 2D 
- is this working on sections or projections, and if so what kind of projections, etc. 
 
Line 137: I don’t think it’s clear that lateral elements directly bind to chromatin, nor that central 
elements bind to lateral elements. Figure 1's legend states that the transverse filaments "connect" 
lateral and central elements, but this is not known and may or may not be a universal feature. 
 
Line 154: “…revealed that the lateral element protein SYCP3 shows a bimodal distribution 
separated by 221.6 ± 6.1 nm (SD) (Fig. 2)” -- but Fig. 2 does not show this value, nor a bimodal 
distribution. 
 
Line 197 refers to U-ExM-induced SC structural breaks shown in Sup. Figure 4, but which panel 
within figure 4 shows the breaks is not mentioned, nor are the breaks highlighted or mentioned 
within the figure; please annotate this so the reader can follow the text. Likewise for lines 200-
201, “pre-expansion labeling resulted in a lower labeling density due to irreversible fluorophore 
destruction during free-radical polymerization (Supplementary Figs. 4 and 5)”, but these figures do 
not point this out, in the figure itself or the legends. 
 
From line 211, "the main difference between centrioles and SCs is their biomolecular composition" 
until line 219, the text unfortunately becomes quite difficult to understand. I think this part should 
be rewritten and placed in the discussion, since it appears to be rationalizing why these results 
were different from previous results with centrioles. 
 
Line 230 -- re: previous dSTORM experiments showing C termini at the lateral elements and N 
termini in the center — it would be good to point out that immunoEM also showed this in the mid-
90's (Liu, J. G. et al. Localization of the N-terminus of SCP1 to the central element of the 
synaptonemal complex and evidence for direct interactions between the N-termini of SCP1 
molecules organized head-to-head. Exp. Cell Res. 226, 11–19 (1996).) 
 
Lines 261-270: if you are using terms like "lateral" and "frontal" to describe the views of the SC, 
please provide an explanatory diagram in an early figure (figure 1 would be a good place) where 
these are defined, otherwise the reader may not understand what is meant. 
 
Lines 275–278: I am not aware of evidence that shows the central element is particularly 
susceptible to competition effects in immunolabeling due to its density; if a reference could be 
provided, please do so; otherwise, if this is speculative, it should not be presented as fact. 
 
Fig. 5e, please indicate the XY pair with arrows (it may not be obvious to everyone). 
 
Several mentions are made of “twisted areas” or “places where the lateral elements twist”; but 
since the lateral elements are twisting everywhere to some extent, it would be good to make sure 
the reader understands that it’s not the case that axes only twist at places that can be observed as 
crossings in the XY plane. 
 
Line 447: "as described under 'proexm protocol'", but there is no such subheading—should be 
written "Protein retention protocol". 
Line 520: “To avoid drift during acquisition a nose-piece stage (IX2-NPS, Olympus) is implemented 
in the microscope.” -- this needs more detail; a catalog number doesn't describe in a reproducible 
way how drift is avoided. 
 
Line 524: "weight per volume" could be more precisely expressed as grams / milliliters (if this is 
the case). 
 
--- 
Peter Carlton, Kyoto University 
 
 
Reviewer #2 (Remarks to the Author): 
 
Review: 
In Zwettler et al., the authors developed an optimized expansion microsopy protocol for visualizing 
the synaptonemal complex. To do so, the authors tested and optimized several expansion 
microscopy variants for both isotropy, labeling density with respect to SC ExM. They demonstrated 
3 color SIM together with an optimized version of MAP, an ExM variant. The manuscript is well 
written, detailed and data from experiments is clear. I think the authors could make a stronger 
point about the significance of the biological discovery, as well as the methodological 
improvements, and how does it apply to those outside of SC biology. 
I think the authors could address the following main points before recommendation for publication 
at Nature Comms: 
 
Major comments: 
Can the authors clarify why the expansion factors used for different versions of the ExM protocol. 
The expansion factor in ExM can be tuned with the crosslinker density, for proper comparison of 
labeling density: all protocols be compared at ~ same expansion factor of 3 between ExM, MAP 
and U-ExM? 
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? 
The authors make several comparisons with EM, stating that MAP-SIM resolves similar structures 
(i.e. SYCP3 fraying). Since MAP-SIM has molecular resolution, as well as multiple colors, have the 
authors been able to discover aspects of the molecular architecture that was previously unknown? 
Minor comment: 
The authors developed a line cut tool and made it broadly available, which is very commendable. 
It’s unclear of computational tool is generally useful for other applications beyond the SC complex. 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
Zwettler et al reports various expansion microsocpy (ExM) methods combined with structured 
illumination microscopy (SIM) to investigate the molecular architecture of the synaptonemal 
complex (SC). The authors developed an image processing software for unbiased generation of 
line profiles from super-resolution images of the synaptonemal complexes. By comparing the 
established structural data from dSTORM, three different expansion methods of ProExM, U-ExM 
and MAP were evaluated in terms of expansion factors and labeling densities. MAP-SIM with the 
best labeling density was used for revealing new structural features of SCs. 
 
Since ExM allows for super-resolution imaging with robust commercial instruments, many 
expansion protocols has been introduced rapidly. However, for ExM to address new, unknown 
structures, it is crucial to precisely quantify the value and uniformity of the expansion factor. This 
paper provides a reference structure and automated software that are suited for providing such 
evaluation platform for ExM. However, the current version of manuscript do not provide sufficient 
supporting data for validating the performance of the software, the isotropic expansion of the MAP 
protocol and their new findings on SC structures from 3-color SIM imaging. For publication, I 
request for more supporting data that I explain in detail as folllws. 
 
1. What are the criteria that “Line Profiler” software choose the area for generating line profiles for 
measuring SC widths and for generating averaged profiles in Supp Figure 1-3 and 5? In other 
words, how did the authors select the white crossing lines in Figs. 4f, Supplementary Fig. 1a-b, 2a-
b and 3a/c? The software output image in Supp Fig. 6 suggests that the software make line 
profiles everywhere along the SC. But, somehow the white lines that I mentioned indicate that 
subsets of line profiles were used to measure peak-to-peak distances and to generate average 
profiles. The selection procedure could impose bias to the statistical results, thus the details should 
be described in the paper. 
 
2. Why do the average values of SYCP3 strand distances from 2-channel and 1-channel analyses in 
Supp Figure 1 and 2 differ so much (580/607 nm vs. 632/635 nm)? Since the sample preparation 
is the same, the expansion factor should be the same. Why did the authors used the value from 1-
channel analysis results to calculate the expansion factor? If 1-channel analysis is more precise or 
more reproducible, evaluate the performances of the two different analyses and explain why their 
results differ. 
 
3. The authors claim “isotropic” expansion by simply showing the images. I cannot judge the 
isotropicity by visual inspection of Fig. 3. ExM papers often show transformation matrix by 
overlapping pre- and post-expansion images. I cannot find such quantitative measures in this 
paper. Or, instead, since the authors developed the image processing software, the distributions of 
SYCP3 strand distances can be used to quantitatively evaluate the isotropicities of proExM, U-ExM 
and MAP at the ultrastructural level. Since the expansion factors differ for proExM, U-ExM and 
MAP, I suggest to normalize each distribution by the corresponding expansion factor for 
comparision of the three expansion protocols. 
 
4. From a single profile example in Fig. 4d, the authors claimed “far more complex distribution of 
the central region proteins than the bimodal distribution”. Simply saying “more complex” is not 
helpful to figure out the differences. Describe more specifically how the distribution differ from 
bimodal. Also, prove the higher complexicity with more data. One example image is not enough. 
 
5. The only obviously new structures in the paper is the bifurcation and fraying of the two SC 
strands in Fig. 5. I am not an expert in meiosis to judge the importance of the findings. The 
authors discussion on these structures sounds descriptive to me. What would be the biological, 
functional implications of these new findings? 
 
Followings are minor comments. 
 
a) The following sentence in the introduction does not make sense. 
“The spreading of the SCs directly onto the surface of the coverslip results in the localization of the 
proteins close to the coverslip post-expansion and allows the use of a water-immersion objective 
for imaging of hydrogels without spherical aberrations.” 
If the sample is placed close to the surface, even oil immersion lens can be used. Did the authors 
mean that high NA lenses can used? 
 
b) In the last paragraph of the introduction, the authors claim the resolution of ExSTORM is 20-30 
nm, which is typical resolution for unexpanded STORM. The ExSTORM paper claims for 10-20 nm 
resolution. Do the authors of this paper have evidences supporting that ExSTORM resolution is 
worse than reported? 
 
c) High-resolution STORM may be limited in two colors. But, three-color DNA-PAINT is possible 
with exchangePAINT and offers even higher resolution than STORM. What would be the 
advantages of 3-color MAP-SIM over 3-color DNA-PAINT? 
 
d) Online Methods, “Immunofluorescence of non-expanded SCs”: What is NGS? Normal goat 
serum? 
 
e) Figure 3. Caption: There is no “g” panel in the figure. Perhaps, the “g” in the caption is typo for 
“f”.