Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles

Article activity feed

1. 

   Version published to 10.7554/elife.79344 on eLife

   Jan 16, 2023
2. 

   eLife

   Oct 25, 2022

   Author Response

   Reviewer #1 (Public Review):

   The authors use the nanobody tools generated in the companion manuscript and have combined them with DNA-Paint oligonucleotide labeling to generate super-resolution images of indirect flight muscles. Using this approach, they could map the precise organization of the different domains from the two giant titin-like fly homologs called Sallimus and Projectin against which the nanobodies had been raised with a precision ranging from 1 nm to 4 nm, depending on the distance between them. They show that in indirect flight muscles the N-ter of Sallimus is located within 50 nm of the Z-disc, and that its C-ter reaches the A-band roughly 100 nm away from the Z-disc. Likewise, they show that the N-ter of Projectin colocalizes with the C-ter of Sallimus at the edge of the A-band, whereas its C-ter is …

   Author Response

   Reviewer #1 (Public Review):

   The authors use the nanobody tools generated in the companion manuscript and have combined them with DNA-Paint oligonucleotide labeling to generate super-resolution images of indirect flight muscles. Using this approach, they could map the precise organization of the different domains from the two giant titin-like fly homologs called Sallimus and Projectin against which the nanobodies had been raised with a precision ranging from 1 nm to 4 nm, depending on the distance between them. They show that in indirect flight muscles the N-ter of Sallimus is located within 50 nm of the Z-disc, and that its C-ter reaches the A-band roughly 100 nm away from the Z-disc. Likewise, they show that the N-ter of Projectin colocalizes with the C-ter of Sallimus at the edge of the A-band, whereas its C-ter is located about 250 nm away in the A-band and 350 nm from the Z-disc. It overall suggests a staggered and linear organization of both proteins with a potential area of overlap spanning 10-12 nm, that Sallimus could bridge the Z-disc to the A-band acting as a ruler, while Projectin should only overlap with 15% of the A-band and possibly a 10 nm of the I-band.

   Thanks for this nice summary of our findings.

   The value of this work comes from its use of advanced technologies (DNA-Paint + superresolution). The biological conclusions confirm and refine earlier and recent papers, especially EM papers and the impressive and very comprehensive JCB paper by Szikora et al in 2020, although the conclusions of the present work differ somewhat from those of Szikora who had predicted that Sallimus does not reach the A-band. That aspect could have been better discussed.

   We have further extended our discussions of the results from Szikora et al. 2020, in particular regarding Sallimus in this revised version.

   Reviewer #2 (Public Review):

   Taking advantage of the high molecular order of the Drosophila flight muscle, Schueder, Mangeol et al. leverage small (<4 nm) original nanobodies, tailored coupling to fluorophores, and DNA-PAINT resolution capabilities, to map the nanoarchitecture of two titin homologs, Sallismus and Projectin.

   Using a toolkit of nanobodies designed to bind to specific domains of the two proteins (described in the companion article "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins" ), Schueder, Mangeol et al position these domains within the sarcomere with <5nm resolution, and demonstrate that the N-ter of Sallismus overlaps with the C-ter of Projectin at the A-band/I-band interface. They propose this architecture may help to anchor Sallismus to the muscle, thus supporting flight muscle function while ensuring muscle integrity.

   This study nicely extends previous work by Szikora et al, and precisely dissect the the sarcomeric geography of Sallismus and Projectin. From these results, the authors formulate specific functional hypotheses regarding the organization of flight muscles and how these are tuned to the mechanical constraints they undergo.

   Although they remain descriptive in essence, the conclusions of the paper are well supported by the experimental results.

   We thank this reviewer for the nice summary of our results.

   Reviewer #3 (Public Review):

   This manuscript by Schueder et al. provides new insight into an important question in muscle biology: how can the smaller titin-like molecules of the much larger sarcomeres of invertebrate muscle perform the same function as the larger titin of vertebrate muscles which have smaller sarcomeres? These functions include the assembly, stability and elasticity of the sarcomere. Using two state of the art methods--nanobodies and DNA-PAINT superresolution microscopy, the authors definitively show that in the highly ordered indirect flight muscle of Drosophila, the elongated proteins Sallimus and Projectin are arranged such that the N-terminus of Sallimus is embedded in the Z-disk, and the C-terminus is embedded in the outer portion of the A-band, and that in this outer portion of the A-band is also embedded the C-terminus of Projectin; thus, if the C-terminus of Sallimus can bind to thick filaments, and/or these overlapping portions of Sallimus and Projectin interact, there would be a linkage of the Z-disk and/or thin filament to the thick filaments to help determine the length and stability of the sarcomere.

   The strengths of this paper include the implementation of nanobody and DNA-PAINT superresolution microscopy for the first time for muscle. The extraordinary 5-10 nm resolution of this method alloiws imaging for definitive localization of the termini of these elongated proteins in the Drosophila flight muscle sarcomere. In addition, the manuscript is well written with sufficient background information and rationale presented, is easy to read, complex new methods are well-described, the figures are of high quality, and the conclusions are well-justified. A minor weakness is that despite the authors demonstrating that the Cterminus of Sallimus is located at the outer edge of the A-band, and that the N-terminus of Projectin is located also in the outer edge of the A-band, the authors provide no data to show whether, for example, these portions of these titin-like molecules interact, or whether Sallimus might interact with thick filaments. Such data would be required to prove their model. However, I can understand that this would require extensive additional study, and the authors have already provided a tremendous amount of data for this first step in supporting the model. Nevertheless, the authors should cite a relevant previous study on the Sallimus homolog in C. elegans called TTN-1, which is also a 2 MDa polypeptide of similar domain organization to at least the large isoforms of Salliums found in fly synchronous muscles. In the study by Forbes et al. (2010), immunostaining, albeit not to the impressive resolution achieved in the present paper, showed that TTN-1 was also localized to the I-band with extension into the outer edge of the A-band. More importantly, that study also showed that "fragment 11/12", Ig38-40, which is located fairly close to the C-terminus of TTN-1 binds to myosin with nanomolar affinity (Kd= 1.5 nM), making plausible the idea that TTN-1 may bind to the thick filament in vivo.

   We thank this reviewer for sharing his enthusiasm about our results and methodology, and also about the way the data are presented. This is one more argument for us to leave a shortened Figure 1 in the PAINT manuscript.

   We are particularly thankful for pointing out the important C. elegans data that we had missed and that, as the reviewer said, perfectly fit with the model we propose for flight muscle (and also the larval muscle data, as the C-term of Sls is the same). Hence, we highlight this paper now in our discussion and compare to our findings.

   Reviewer #4 (Public Review):

   This manuscript reports combining recently developed and described in the accompanying paper nanobodies against Sallimus and Projectin with DNA-Paint technology that allows super-resolution imaging. Presented data prove that such a combination provides a powerful system for imaging at a nano-scale the large and protein-dense structures such as Drosophila flight muscle. The main outcome is the observation that in flight muscle sarcomeres Salimus and Projectin overlap at the I/A band border. This was elegantly achieved using double color DNA-Paint with Sls and Projectin nanobodies.

   We thank the reviewer for appreciating the quality of our work.

   Overall, as it stands, this manuscript even if of high technological value, remains entirely descriptive and short in providing new insights into muscle structure and architecture. The main finding, an overlap between short Sls isoform and Proj in flight muscle sarcomeres, is redundant with the author's observation (described in the companion paper "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins") that in larval muscles expressing a long Sls isoform, Sls and Proj overlap as well.

   Alternatively, combination of Sls and Proj nanobodies with DNA-Paint represents an interesting example of technological development that could strengthen the accompanying nanobodies toolkit manuscript.

   Every structural paper reports the structure and is thus by definition descriptive. This is the aim of our manuscript. We do not think that the other nanobody resource paper reports an overlap of Sls and Projectin in the larvae. To resolve such a possible overlap, super resolution would be needed. The other paper does report that larval Sls isoform is dramatically stretched, more than 2 µm, and that Projectin is decorating the thick filament, likely in an oriented manner. If N-term of Projectin overlaps with C-term of Sallimus in this muscle is an open question that needs DNA-PAINT imaging of larval muscle. This requires a TIRF setting that is technically not trivial to achieve for larval muscle and hence has not been done by anybody.

   Read the original source
3. 

   eLife

   Oct 25, 2022

   eLife assessment

   The manuscript is of broad interest in the field of muscle physiology and structure. The authors developed nanobodies against different domains of the giant Drosophila proteins Sallimus and Projectin, which are titin homologs, and used them to define their organization along sarcomeres of distinct fly muscles. This is an important contribution to understand the functional architecture of the muscle; it suggests that in invertebrates two proteins fulfil the role of the vertebrate titin in bridging the A-band and the I-band.

   This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2022.04.13.488177v1

   Read the original source
4. 

   eLife

   Oct 25, 2022

   Reviewer #1 (Public Review):

   The authors use the nanobody tools generated in the companion manuscript and have combined them with DNA-Paint oligonucleotide labeling to generate super-resolution images of indirect flight muscles. Using this approach, they could map the precise organization of the different domains from the two giant titin-like fly homologs called Sallimus and Projectin against which the nanobodies had been raised with a precision ranging from 1 nm to 4 nm, depending on the distance between them. They show that in indirect flight muscles the N-ter of Sallimus is located within 50 nm of the Z-disc, and that its C-ter reaches the A-band roughly 100 nm away from the Z-disc. Likewise, they show that the N-ter of Projectin colocalizes with the C-ter of Sallimus at the edge of the A-band, whereas its C-ter is located about 250 …

   Reviewer #1 (Public Review):

   The authors use the nanobody tools generated in the companion manuscript and have combined them with DNA-Paint oligonucleotide labeling to generate super-resolution images of indirect flight muscles. Using this approach, they could map the precise organization of the different domains from the two giant titin-like fly homologs called Sallimus and Projectin against which the nanobodies had been raised with a precision ranging from 1 nm to 4 nm, depending on the distance between them. They show that in indirect flight muscles the N-ter of Sallimus is located within 50 nm of the Z-disc, and that its C-ter reaches the A-band roughly 100 nm away from the Z-disc. Likewise, they show that the N-ter of Projectin colocalizes with the C-ter of Sallimus at the edge of the A-band, whereas its C-ter is located about 250 nm away in the A-band and 350 nm from the Z-disc. It overall suggests a staggered and linear organization of both proteins with a potential area of overlap spanning 10-12 nm, that Sallimus could bridge the Z-disc to the A-band acting as a ruler, while Projectin should only overlap with 15% of the A-band and possibly a 10 nm of the I-band.

   The value of this work comes from its use of advanced technologies (DNA-Paint + super-resolution). The biological conclusions confirm and refine earlier and recent papers, especially EM papers and the impressive and very comprehensive JCB paper by Szikora et al in 2020, although the conclusions of the present work differ somewhat from those of Szikora who had predicted that Sallimus does not reach the A-band. That aspect could have been better discussed.

   Read the original source
5. 

   eLife

   Oct 25, 2022

   Reviewer #2 (Public Review):

   Taking advantage of the high molecular order of the Drosophila flight muscle, Schueder, Mangeol et al. leverage small (<4 nm) original nanobodies, tailored coupling to fluorophores, and DNA-PAINT resolution capabilities, to map the nanoarchitecture of two titin homologs, Sallismus and Projectin.

   Using a toolkit of nanobodies designed to bind to specific domains of the two proteins (described in the companion article "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins" ), Schueder, Mangeol et al position these domains within the sarcomere with <5nm resolution, and demonstrate that the N-ter of Sallismus overlaps with the C-ter of Projectin at the A-band/I-band interface. They propose this architecture may help to anchor Sallismus to the muscle, thus supporting flight muscle …

   Reviewer #2 (Public Review):

   Taking advantage of the high molecular order of the Drosophila flight muscle, Schueder, Mangeol et al. leverage small (<4 nm) original nanobodies, tailored coupling to fluorophores, and DNA-PAINT resolution capabilities, to map the nanoarchitecture of two titin homologs, Sallismus and Projectin.

   Using a toolkit of nanobodies designed to bind to specific domains of the two proteins (described in the companion article "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins" ), Schueder, Mangeol et al position these domains within the sarcomere with <5nm resolution, and demonstrate that the N-ter of Sallismus overlaps with the C-ter of Projectin at the A-band/I-band interface. They propose this architecture may help to anchor Sallismus to the muscle, thus supporting flight muscle function while ensuring muscle integrity.

   This study nicely extends previous work by Szikora et al, and precisely dissect the the sarcomeric geography of Sallismus and Projectin. From these results, the authors formulate specific functional hypotheses regarding the organization of flight muscles and how these are tuned to the mechanical constraints they undergo.

   Although they remain descriptive in essence, the conclusions of the paper are well supported by the experimental results.

   Read the original source
6. 

   eLife

   Oct 25, 2022

   Reviewer #3 (Public Review):

   This manuscript by Schueder et al. provides new insight into an important question in muscle biology: how can the smaller titin-like molecules of the much larger sarcomeres of invertebrate muscle perform the same function as the larger titin of vertebrate muscles which have smaller sarcomeres? These functions include the assembly, stability and elasticity of the sarcomere. Using two state of the art methods--nanobodies and DNA-PAINT super-resolution microscopy, the authors definitively show that in the highly ordered indirect flight muscle of Drosophila, the elongated proteins Sallimus and Projectin are arranged such that the N-terminus of Sallimus is embedded in the Z-disk, and the C-terminus is embedded in the outer portion of the A-band, and that in this outer portion of the A-band is also embedded the …

   Reviewer #3 (Public Review):

   This manuscript by Schueder et al. provides new insight into an important question in muscle biology: how can the smaller titin-like molecules of the much larger sarcomeres of invertebrate muscle perform the same function as the larger titin of vertebrate muscles which have smaller sarcomeres? These functions include the assembly, stability and elasticity of the sarcomere. Using two state of the art methods--nanobodies and DNA-PAINT super-resolution microscopy, the authors definitively show that in the highly ordered indirect flight muscle of Drosophila, the elongated proteins Sallimus and Projectin are arranged such that the N-terminus of Sallimus is embedded in the Z-disk, and the C-terminus is embedded in the outer portion of the A-band, and that in this outer portion of the A-band is also embedded the C-terminus of Projectin; thus, if the C-terminus of Sallimus can bind to thick filaments, and/or these overlapping portions of Sallimus and Projectin interact, there would be a linkage of the Z-disk and/or thin filament to the thick filaments to help determine the length and stability of the sarcomere.

   The strengths of this paper include the implementation of nanobody and DNA-PAINT super-resolution microscopy for the first time for muscle. The extraordinary 5-10 nm resolution of this method allows imaging for definitive localization of the termini of these elongated proteins in the Drosophila flight muscle sarcomere. In addition, the manuscript is well written with sufficient background information and rationale presented, is easy to read, complex new methods are well-described, the figures are of high quality, and the conclusions are well-justified. A minor weakness is that despite the authors demonstrating that the C-terminus of Sallimus is located at the outer edge of the A-band, and that the N-terminus of Projectin is located also in the outer edge of the A-band, the authors provide no data to show whether, for example, these portions of these titin-like molecules interact, or whether Sallimus might interact with thick filaments. Such data would be required to prove their model. However, I can understand that this would require extensive additional study, and the authors have already provided a tremendous amount of data for this first step in supporting the model. Nevertheless, the authors should cite a relevant previous study on the Sallimus homolog in C. elegans called TTN-1, which is also a 2 MDa polypeptide of similar domain organization to at least the large isoforms of Salliums found in fly synchronous muscles. In the study by Forbes et al. (2010), immunostaining, albeit not to the impressive resolution achieved in the present paper, showed that TTN-1 was also localized to the I-band with extension into the outer edge of the A-band. More importantly, that study also showed that "fragment 11/12", Ig38-40, which is located fairly close to the C-terminus of TTN-1 binds to myosin with nanomolar affinity (Kd= 1.5 nM), making plausible the idea that TTN-1 may bind to the thick filament in vivo.

   Read the original source
7. 

   eLife

   Oct 25, 2022

   Reviewer #4 (Public Review):

   This manuscript reports combining recently developed and described in the accompanying paper nanobodies against Sallimus and Projectin with DNA-Paint technology that allows super-resolution imaging. Presented data prove that such a combination provides a powerful system for imaging at a nano-scale the large and protein-dense structures such as Drosophila flight muscle. The main outcome is the observation that in flight muscle sarcomeres Salimus and Projectin overlap at the I/A band border. This was elegantly achieved using double color DNA-Paint with Sls and Projectin nanobodies.

   Overall, as it stands, this manuscript even if of high technological value, remains entirely descriptive and short in providing new insights into muscle structure and architecture.
   The main finding, an overlap between short Sls …

   Reviewer #4 (Public Review):

   This manuscript reports combining recently developed and described in the accompanying paper nanobodies against Sallimus and Projectin with DNA-Paint technology that allows super-resolution imaging. Presented data prove that such a combination provides a powerful system for imaging at a nano-scale the large and protein-dense structures such as Drosophila flight muscle. The main outcome is the observation that in flight muscle sarcomeres Salimus and Projectin overlap at the I/A band border. This was elegantly achieved using double color DNA-Paint with Sls and Projectin nanobodies.

   Overall, as it stands, this manuscript even if of high technological value, remains entirely descriptive and short in providing new insights into muscle structure and architecture.
   The main finding, an overlap between short Sls isoform and Proj in flight muscle sarcomeres, is redundant with the author's observation (described in the companion paper "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins") that in larval muscles expressing a long Sls isoform, Sls and Proj overlap as well.
   Alternatively, combination of Sls and Proj nanobodies with DNA-Paint represents an interesting example of technological development that could strengthen the accompanying nanobodies toolkit manuscript.

   Read the original source
8. 

   Version published to 10.1101/2022.04.14.488306v1 on bioRxiv

   Apr 15, 2022