Peer review of: Deconstructing sarcomeric structure–function relations in titin-BioID knock-in mice (2021)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 


This manuscript by Rudolph et. al. describes the generation and use of a mouse model in which BioID 
has been knocked into the titin gene to identify protein-protein associations of this large structural 
protein that functions in muscle sarcomeres. These mice were used for BioID pulldowns using two 
different methods, one that identified sites of protein biotinylation to map proximities of protein 
domains with high resolution and another that pulled down all biotinylated proteins to map protein 
proximities. These pulldowns were performed from both heart and quadricep muscle and compared 
between newborn and adult tissues. Overall the generation of the mice appears well conceived and 
the experimental design was well thought out. There appears to be some confusion by the authors 
as to the interpretation of the conventional BioID pulldowns as somehow being able to isolate intact 
and stable protein complexes, in comparison to the method to capture biotinylated peptides which 
yielded far fewer detected proteins. Overall, this is a basic proof of principle application of BioID in a 
mouse where the ligase has been fused to an endogenously expressed protein. The identified 
proteins associated with titin should be of value to investigators in that field. 

The duration of biotin administration should be mentioned in the methods. 


Consider citing PMID 29249144 along with the Carr paper for the anti-biotin method since they were 
effectively independently co-developed with one coming out just before the other. 


Could the authors show a full Western blot probed for biotinylated protein to show the extent of 
biotinylation and distribution of those proteins by MW. 


The authors say: ‘To extend our approach of in vivo colocalization proteomics from Z-disc titin to the 
sarcomere, we used an adapted streptavidin based pull-down protocol eliminating the initial trypsin 
digest to enrich entire sarcomere complexes.’ What it looks like they are describing is in fact a 
normal BioID pulldown experiment that captures proteins biotinylated by BioID-titin. With the 
buffers used in the lysis and washing there should be no remaining protein-protein interactions, or 
complexes (500mM salts, substantial detergents, etc.). These conditions are designed to denature 
proteins allowing just the biotinylated ones to be selectively isolated. My suspicion is that the anti-
biotin pulldown of tryptic peptides that only identified 14 proteins was simply very inefficient and is 
only detecting the most abundant biotinylated peptides. It doesn’t change the results for those anti-
biotin AB experiments, in that it can inform on the relative proximities of these proteins to the 
ligase, but it doesn’t mean that those were the only proteins biotinylated. Both the Carr and the 
Pandey (PMID 29249144) papers showed that there was relatively similar detection of proteins with 
the conventional pulldown and the anti-biotin method with considerable overlap in those proteins 
detected. The discrepancy here suggests a technical issue with the capture of biotinylated peptides. 


The authors should acknowledge that all BioID experiments should have a control, typically ligase-
alone, or something similar to subtract proteins that are either non-specifically pulled down and/or 
proteins with an affinity to the ligase itself, of which there are many. Given the cost and effort to 
generate these mice, the lack of such a control can be forgiven; however, this issue must be 
addressed to qualify the limitations of the results. 


The authors need to provide the MS result in a excel file with the identities of the proteins and 
relative abundance as is conventional for these types of studies. 


In the methods there is mention of ‘Global proteome of neonatal and adult mouse tissue by LC-MS’. 
What is this is reference to? 



 

Reviewer #2 (Remarks to the Author): 

 

The manuscript titled “Deconstructing sarcomeric structure/function relations in titin-BioID knock-in 
mice” by Rudolph et al. provides an excellent resource for the interactome or signalosome of 
striated muscle sarcomere. And it has the potential to enable further studies with BioID in the 
muscle field following this proof-of-concept work. The authors confirm that the knock-in mice are in 
general healthy and therefore a suitable model to dissect even transient protein-protein interactions 
at the Z-disc in the living animal. This elegant approach harbours plenty of information for functional 
follow-up studies and thereby is interesting for a wider audience in the muscle field and – regarding 
the method – also beyond. 

The authors aim to connect the comprehensive biochemical work on heart and quadriceps in the 
adult and neonatal mouse to achieve a better understanding of structure-function relationship of 
titin with other proteins at striated muscle sarcomere. The biochemical data is an excellent source 
providing information about (potential) interactions on the protein level – which should dominantly 
happen at the z-disc (although the free titin pool can also contribute). These interactions can be 
either transient or between well-known and stable interaction partners. Especially, the potential to 
identify novel (transient) interaction partners could be great value also for future studies in the 
muscle field and other fields. On the other hand, the authors need to pay more attention to present 
the data completely and convincingly, incl. data analysis and data presentation, in particular while 
establishing the relative positioning of various sarcomeric proteins investigated here. The comments 
in detail as following: 


The authors try to support their biochemical findings by immunofluorescence microscopy employing 
super-resolution STED microscopy. To achieve a convincing and balanced experimental support of 
their favoured explanations outlined in the manuscript, they should present especially their STED 
imaging data more detailed and include additional controls. They should also consider adding 
additional experiments supporting their favourite models of sarcomere interaction and muscle 
function. 

In detail: 

The authors should provide more information about the STED imaging in the materials and methods 
section: 

- They state twice (page 19 and 20) that the method “has been described” elsewhere but do not 
provide any reference. The references should be added and the authors might want to consider to 
briefly outline the experimental procedure to avoid confusion, e.g. about the thickness of the tissue 
sections/slices used, STED mode used (2D or 3D – see also below) etc. 

- In the materials and methods, the author mentioned that “imaging was performed with a 3D-STED 
microscope” However, the pixel sizes used for image acquisition would be optimal for so-called 2D-
STED. Based on this, I assume that 2D-STED has been used and for my interpretation of the results. 

General comment to fluorescence representation in all figures: 

- The size of the images is rather small and capturing a large sample area, making it hard if not 
impossible for the reader to confirm the described localization pattern in the text. The authors have 
reserved a lot of space for depicting graphs of proteomic data, which could also be presented in a list 
or a table, while the key imaging-experiments for validating the screen are squeezed into very 
limited space and provide no real chance for the reader to see the confirmation but only to believe 
it. The images should be enlarged and panels displaying multiple region of interested be shown at 
larger magnification (zoom-in). For example, In Fig. 1f or h (see also image attached). Here, after 
zooming in on a screen one can really see that there could be indeed three lines of titin and the 
traces become more believable. In addition, for each image, individual channel images and then a 
merge should be presented. Panels were half the image is just one channel and the other half is a 
merge can be very misleading (see Fig. 1 f and h. 

- For each multi-colour image, at least one, but better multiple line profiles should be displayed for 
all fluorescent markers. 

- The authors should indicate in the materials and methods section, which fluorophores correspond 
to which colour code in the figures (e.g. if Star 580 is always encoded in green). 

- Stating partial co-localization can be a dangerous route. Therefore, it is very important to provide 
the reader with all details and control experiments. The authors should also consider that the 
interaction could be transient or the protein be biotinylated by the non-integrated titin pool and 
thereby no co-localization would be detectable in immunofluorescence. The current presentation of 
the imaging data does neither confirm nor exclude partial-co-localization of Myosin / MyH8, Pgam2 
and Nebl (see also per figure comments below). 

A more extensive (semi-) quantification of the signals (e.g. by averaging multiple line profile on 
different samples and displaying SD or SEM) would also improve the manuscript. 

 

Detailed comments per figure: 

- Fig. 1 f-i: Line Profiles should be shown for both colours (see also above). Both channels should be 
first displayed separately and then with the merge. 

- Fig. 2 h/j: The authors should adhere to the general practice to draw line profiles in a consistent 
manner. This is usually perpendicular to the structure of interest, here the biotinylation region. 

- Ext. Data Fig. 2: k-o partial co-localization is hard to confirm by the reader due to the small size of 
the images and lack of line profiles (see general comments above). Furthermore, in k, the Nebulette 
signal at the sarcolemma is saturated and preventing to estimate the fraction of protein at the z-
disk. The authors might want to clarify this by showing non-normalized line profiles. Signal crosstalk 
between the STED dyes used in this study has been observed on other occasions when the signal 
intensity was substantially stronger in one channel than the other. The authors should briefly clarify 
the used fluorophore combination and add the respective controls to the figure (e.g. by presenting 
representative single staining of the structures imaged with the same microscope settings). 

In my opinion, the authors have undertaken a great task of localising sarcomeric proteins with 
advanced light microscopy, and this can be really helpful for the field. To establish precisely the co-
localization pattern and degree of overlap (and make this easily visible to all readers), images 
displaying the biotinylation band and a immunostaining for a protein that resides 

a) Exclusively outside the z-disc 

b) Exclusively at the z-disk and 

c) are present (to varying degree) in both regions 
should be added. 

 

A relative “sarcomeric map” should be made (see also Kelu et al., Dev Biol, 2017, 
https://doi.org/10.1016/j.ydbio.2017.03.031; Figure 9) to support the reader with the relative 
position of any protein that was measured. 

 

Although, this work has the full potential to become pioneering work. The currently included 
experiments and especially the data presentation provokes the feeling of being partially unfinished 
and causes unnecessary ambiguity. In my opinion, additional experiments could strengthen, the 
overall message of this work and I would like to urge the authors to consider including the following 
experimental methodologies: 

 

1) To support the biochemical data further, a super-resolution immune-fluorescence mapping of 
titin structure by employing antibody directed against various Ig domains of titin (that can possibly 
be resolved by STED) would be more than supportive for the findings presented. This could etsablish 
a better overview of the confirmation of titin in stretched and non-stretched muscle and provide 
additional evidence for the biotinylation pattern of titin. 

 

2) A mapping of myosin in respective to the biotinylation band using different antibodies recognizing 
the rod, the globular domain and the light chains in non-stretched and in stretched muscle should 
provide a detailed view to what extend the myosin is indeed coming close or penetrating the z-disc. I 
can imagine that antibodies recognizing the light chain or the globular domain could provide a better 
and more convincing overlap with the biotinylation band. In addition, this would remove ambiguity 
that the biotinylation band found ~200nm away from the z-disc in the stretched muscle is formed by 
other proteins than myosin. 

 

3) Single muscle fibres have been shown to provide high quality STED images. The authors might 
want to extend their study using these kind of samples. 
(https://www.ahajournals.org/doi/pdf/10.1161/CIRCRESAHA.112.274530) 

 

 

Minor comments but important aspects of the manuscript that can be changed for the ease of 
understanding of a broader audience or non-titin specialist skeletal muscle biologists: 

 

1) Throughout the manuscript, the supplementary figures should be named as “supplementary” not 
as “extend figures” or can be presented the other way around but consistently 

2) Page 5 it should be traces 1g vs i NOT 1e vs i 

3) Please mention if an image is confocal, see Fig 1c 

4) Page 6, the authors reported to have found “14” biotinylated proteins in the main text and “15” in 
the extended Data Fig.2 legend. 

5) Supplementary Table 2: Neonatal Quad vs Neonatal Heart, there is a significant number of 
collagen proteins missing from Neonatal Quad, even though collagens have been shown to be 
essential for skeletal muscle development. This should be discussed and what could this mean in 
terms of the developmental differences between the two muscle types? 

6) Page 10: Second paragraph “As we integrate colocalization information over time”: it is not 
necessarily clear if author have done any time lapse microscopy or want to refer to their 
biotinylation approach. 


Marko Lampe with substantial support by 

Muzamil Majid Khan 


 

Reviewer #3 (Remarks to the Author): 

 

The authors present a very interesting approach for testing protein-protein interactions in the 
cardiac and skeletal muscle sarcomere. By generating a BioID mouse with a biotin-ligase located in 
the Z-disk region of titin the authors are now able to monitor titin interactions with highly improved 
spatio-temporal resolution, compared to previous approaches. 

The experiments are well-performed, statistical analyses are appropiate, description of the methods 
is short but precise. 

Key findings of the paper are: 

- identification of 478 sarcomeric or sarcomere associated proteins, which is much more than 
suggested before. 

- identification of titin Ig8/9 as putative hairpin forming structures that may influence titin-based 
elastic properties of the sarcomere 

- confirmation of previous hypotheses suggesting Z-disk penetration by myosin filaments during 
contraction/relaxation cycle 

- Description of titin as a dynamic signaling hub for signal transduction and metabolism, depending 
on developmental state. 

The manuscript is packed with excellent original data and provides extensive supplementary 
material. Some of the data are confirmatory, others really novel and exciting. 

Description and interpretation of these large data sets in a comprehensive manner and for a large 
readership is indeed challenging. The reader is overwhealmed by the different data that range from 
general protein interaction studies, structural titin-spring studies to developmental changes in 
metabolism. Not to mention the data from proteome analyses. 

At some points the selection of data shown in the graphs seems random, e.g. Figure 2. Here, 
biotinylated metabolic enzymes are presented, including Ldb3, Phtf1 and Pgam2, but most of the 
proteins shown are not really discussed later in the manuscript. 

For the reader it is very hard to follow all these different lines of evidences without getting lost in 
details of the figures. One suggestion could therefore be to focus the manuscript body to the main 
findings and move some of the detailed graphs to the supplement or to an independent manuscript. 

Maybe then it would be possible to go into more detail when explaining e.g. the titin hairpin theory.I 
think this is really exciting!! 

I was a bit lost when looking for the additional subset of identified SR-titin interactions, mentioned 
on page 7. This could also be described in more detail. 


I also feel that a few sentences on the principle of integration of a BioID should be added to the 
introductory section. 


However, the discussion is really well structured and very well written ans thus helps understanding 
the different findings of the manuscript. 


Martina Krüger