A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins
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The work describes the generation of novel reagents, nanobodies, which are single molecule antibodies from alpacas, which the authors raised against specific domains of two giant fly muscle proteins called Sallimus and Projectin. These nanobodies, combined with the so-called DNA-Paint approach, enabled the authors to reach an unprecedented spatial resolution and define the position of those domains. Thereby, the authors could propose a model for the organization and extent of those proteins along muscle sarcomeres.
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Abstract
Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.
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Author Response
Reviewer #1 (Public Review):
The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce …
Author Response
Reviewer #1 (Public Review):
The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce the organization of the epitopes in different muscle types and propose a model for Sallimus and Projectin arrangement in muscles, including in larvae which are difficult to label with traditional antibodies due to their impermeable chitin skeleton. Finally, they could fuse the gene encoding one of the nanobodies to the open reading frame of NeonGreen and express the corresponding fusion protein in animals to use the probe in FRAP assays.
The work is very well performed and convincing. However, given its significant redundancy in terms of biological conclusions with the companion study "Nanobodies combined with DNAPAINT super-resolution reveal a staggered titin nano-architecture in flight muscles" by the same authors, and other published papers, I recommend the authors further prove the use of their nanobodies in live assays. In particular, the authors should test whether they can use the nanobodies to induce protein degradation either permanently or conditionally.
Thanks for this nice summary of our findings. We have now extended the analysis of the Nanobody-NeonGreen fusion expressing larval muscles and provide first proof of principle analysis of new fly strains that we generated that contain Sls-Nano2 or Sls-Nano42 nanobodies fused to a degradation signal. These induce lethality of the animals suggesting that Sls protein is partially non functional. We verified this by providing quantitative stainings of various Sls epitopes in these muscles suggesting that Sls is not fully degraded but rather partially modified in the Sls-Nano-deGrad expressing muscle fibers. These will be interesting tools to study Sls function during sarcomere homeostasis.
Reviewer #2 (Public Review):
The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectinspecific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of homology of Drosophila Sallimus and Projectin with related proteins in other species is not discussed. Whether the nanobodies reported here would be useful in other (closely related?) species, therefore, remains to be established. For those studying muscle biology in Drosophila, the nanobodies described here will be publicly available as cDNAs. Ease of production implies a readily shared and standardized resource for the field.
We thank this reviewer for appreciating that our Sallimus and Projectin nanobodies are useful. We now have extended the collection even further, including anti-Obscurin, αActinin and Zasp52 nanobodies, the latter two will also be useful for researcher studying other tissues, in particular Drosophila epithelial tissues. As always in the Drosophila field, all the here generated fly strains and plasmids will be made easily available to the community by placing them in stock centers or shipping them to the laboratories directly. As indicated, also the plasmids will be deposited at Addgene.
Further characterization of these nanobodies by biochemical methods such as immunoblotting would be challenging, given the size of the target proteins. In view of the technical nature of this manuscript, the authors should perhaps critically discuss the distinction between bulky GFP tags versus the much smaller epitope tags and the nanobodies that recognize them, although this was covered in a recent eLife paper from the Perrimon lab. Insertion of small tags, in conjunction with nanobodies that recognize them, would be less perturbing than the much bulkier GFP tag and lend itself to genome-wide applications. Creating nanobodies uniquely specific for each protein encoded in the Drosophila genome is not realistic, and the targeted approach deployed here is obviously valuable.
We are discussing the drawbacks of solely relying on GFP nanobodies, which requires GFP tagged proteins to be available and being functional. In particular for the sarcomeric proteins this is often not the case. We also cite the Perrimon paper, which was just published as we prepared this manuscript. We would like to point out to this reviewer that even tagging with a small epitope tag is considerable work in Drosophila and that the Perrimon paper, on which this reviewer is an author, does describe only two endogenously tagged genes with a nanotag (histone H2Av and Dilp2) the other genes described were expressed from a UAS source or in cell culture. We show here 22 nanobodies against 11 target epitopes.
Nanobodies recognise typically folded epitopes and are rather unlikely to work in immunoblotting.
The authors apply two different approaches to characterize the newly generated Nanobodies: more or less conventional immunohistochemistry with fluorescently labeled nanobodies, and in vivo expression of nanobodies fused to the fluorescent neongreen protein. The superiority of nanobodies in terms of tissue penetration has been shown by others in a direct comparison of intact fluorescently labeled immunoglobulins versus nanobodies. The authors state that in vivo labeling with nanobody fusions "thus far was done only with nanobodies against GFP, mCherry or short epitope tags." There is no fundamental difference between these recognition events and what the authors report for their Sallimus and Projectin-specific reagents. The section that starts at line 304 is thus a little bit of a 'straw man'. There is no reason to assume that a nanobody that recognizes a muscle protein would behave differently than a nanobody that would recognize that same protein (or another) when epitope- or GFP-tagged. What might be interesting is to examine the behavior of these muscle-specific nanobodies in the course of muscle contraction/relaxation: are there conformational alterations that promote dissociation of bound nanobodies? Do different nanobodies display discrete behavior in this regard? The manuscript is silent on how muscles behave in live L3 larvae. The FRAP experiment seems to suggest that not much is happening, but the text refers to the contraction of larval sarcomeres from 8.5 µM to 4.5 µM. Does the in vivo expressed nanobody remain stably bound during this contraction/relaxation cycle? What about the other nanobodies reported in this manuscript? Since the larval motion was reduced by exposure to diethylether, have the authors considered imaging the contractive cycle in the absence of such exposure?
We appreciate the expert knowledge about nanobodies by this reviewer. However, nanobodies were not extensively applied in Drosophila tissues. Hence, we believe it is important to characterise their penetration in stainings and compare them carefully to antibodies. Such, the Drosophila reader will be aware of their advantages.
We have now also included more data on the larval muscle morphology in the nanobody expressing muscles. Their morphology is normal. As larvae move around extensively all the time, the binding of the nanobodies to the target must be stable, otherwise it would not be bound when we fix them or anesthetize them. However, we have not attempted to image them at high resolution while crawling freely. From quantifying the crawling speed (about 1.5 mm per second, see Figure 9 S1) we hope this reviewer appreciates that high resolution imaging of sarcomeres in freely crawling larvae is highly non trivial.
Given that the nanobodies bind well-folded epitopes with low picomolar dissociations constants, it is hard to imagine that conformational changes of the target would dissociate them. The nanobody would stabilise the recognised conformation by a ΔG of ≈60 KJ/ mole, and we would not expect that the chosen domains undergo major conformational changes.
Reviewer #3 (Public Review):
Loreau et al. have presented a well-written manuscript reporting clever, original work taking advantage of fairly new biotechnology - the generation and use of single chain antibodies called nanobodies. The authors demonstrate the production of multiple nanobodies to two titin homologs in Drosophila and use these nanobodies to localize these proteins in several fly muscle types and discover interesting aspects of the localization and span of these elongated proteins in the muscle sarcomere. They also demonstrate that one of these single chain antibodies can be expressed in muscle fused to a fluorescent protein to image the localization of a segment of one of these giant proteins called Sallimus in muscle in a live fly. Their project is well-justified given the limitations of the usual approaches for localizing and studying the dynamics of proteins in the muscle of model organisms such as the possibility that GFP tagging of a protein will interfere with its localization or function, and poor penetration of large IgG or IgM antibodies into densly packed structures like the sarcomere after fixation as compared to smaller nanbodies.
They achieved their goals consistent with the known/expected properties of nanobodies: (1) They demonstrate that at least one of their nanobodies binds with very high affinity. (2) They bind with high specificity. (3) The nanobodies show much better penetration of fixed stage 17 embryos than do conventional antibodies.
They use their nanobodies mostly generated to the N- and C-terminal ends of Sallimus and Projectin to learn new information about how these elongated proteins span and are oriented in the sarcomere. For example, in examining larval muscles which have long sarcomeres (8.5 microns), using nanobodies to domains located near the N- and C-termini, they show definitively that the predicted 2.1 MDa protein Sallimus spans the entire I-band and extends a bit into the A-band with its N-terminus embedded in the Z-disk and C-terminus in the outer edge of the A-band. Using a similar approach they also show that the 800 kDa Projectin decorates the entire myosin thick filament except for the H-zone and M-line in a polar orientation. Their final experiment is most exciting! They were able to express in fly larval muscles a nanobody directed to near the N-terminus of Sallimus fused to NeonGreen and show that it localizes to Z-disks in living larvae, and by FRAP experiments demonstrate that the binding of this nanobody to Sallimus in vivo is very stable. This opens the door to using a similar approach to study the assembly, dynamics, and even conformational changes of a protein in a complex in a live animal in real time.
We thank this reviewer for appreciating the quality and impact of our approach and the our obtained results.
There are only a few minor weaknesses about their conclusions: (1) They should note that in fact their estimate of the span of Sallimus could be an underestimate since their Nano2 nanobody is directed to Ig13/14 so if all of these 12 Ig domains N-terminal of their epitope were unwound it would add 12 X 30 nm = 360 nm of length, and even if unwound would add about 50 nm of length.
We are discussing the length contribution of the 12 Ig domains now more extensively in the DNA PAINT super-resolution paper, however not in this resource paper as the 50 nm difference was not resolved with the confocal microscopy applied here to the larval muscle sarcomere.
(2) They discuss how Sallimus and Projectin are the two Drosophila homologs of mammalian titin, however, they ignore the fact that there is more similarity between Sallimus and Projectin to muscle proteins in invertebrates. For example, in C. elegans, TTN-1 is the counterpart of Sallimus, and twitchin is the counterpart of Projectin, both in size and domain organization. The authors present definitive data to support Figure 9, their nice model for a fly larval sarcomere but fail to point out that this model likely pertains to C. elegans and other invertebrates. In Forbes et al. (2010) it was shown that TTN-1, which can be detected by western blot as ~2 MDa protein and using two polyclonal antibodies spans the entire Iband and extends into the outer edge of the A-band, very similar to what the authors here have shown, more elegantly for Sallimus. In addition, several studies have shown that twitchin (Projectin) does not extend into the M-line; the M-line is exclusively occupied by UNC-89, the homolog of Obscurin.
We thank this reviewer for pointing out the important C. elegans literature that we have now included in this revised manuscript. We apologise for initially omitting them. They are indeed highly relevant.
Reviewer #4 (Public Review):
Authors report the generation and characterisation of several nanobodies for giant Drosophila sarcomeric proteins, Sallimus and Projectin the functional orthologs of titin. They describe an efficient pipeline that could potentially help in designing and producing nanobodies for other proteins. There are several advantages to using nanobodies in comparison to conventional antibodies and the authors nicely demonstrate that the generated nanobodies allow to precisely map subcellular localisation and even the protein orientation in the case of Projectin. They also show that small nanobody molecules have superior penetration and labelling efficiencies with respect to classical antibodies. Finally, the authors select one of the nanobodies to test whether it will efficiently detect native proteins in living tissue. They confirm that Sls-Nano2NeoGreen binds Sls in vivo in muscles of temporarily immobilized 3rd instar larva allowing to reveal sarcomeric Sls pattern and to demonstrate by FRAP experiments that Sls does not exchange during a short time period.
This work is of significant value to a large audience. It provides a clear and precise pipeline for the generation of efficient nanobodies, which are invaluable tools of modern biology.
We thank this reviewer for expressing strong support for our manuscript and appreciating its value for a large readership.
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Reviewer #4 (Public Review):
Authors report the generation and characterisation of several nanobodies for giant Drosophila sarcomeric proteins, Sallimus and Projectin the functional orthologs of titin. They describe an efficient pipeline that could potentially help in designing and producing nanobodies for other proteins. There are several advantages to using nanobodies in comparison to conventional antibodies and the authors nicely demonstrate that the generated nanobodies allow to precisely map subcellular localisation and even the protein orientation in the case of Projectin. They also show that small nanobody molecules have superior penetration and labelling efficiencies with respect to classical antibodies. Finally, the authors select one of the nanobodies to test whether it will efficiently detect native proteins in living tissue. …
Reviewer #4 (Public Review):
Authors report the generation and characterisation of several nanobodies for giant Drosophila sarcomeric proteins, Sallimus and Projectin the functional orthologs of titin. They describe an efficient pipeline that could potentially help in designing and producing nanobodies for other proteins. There are several advantages to using nanobodies in comparison to conventional antibodies and the authors nicely demonstrate that the generated nanobodies allow to precisely map subcellular localisation and even the protein orientation in the case of Projectin. They also show that small nanobody molecules have superior penetration and labelling efficiencies with respect to classical antibodies. Finally, the authors select one of the nanobodies to test whether it will efficiently detect native proteins in living tissue. They confirm that Sls-Nano2NeoGreen binds Sls in vivo in muscles of temporarily immobilized 3rd instar larva allowing to reveal sarcomeric Sls pattern and to demonstrate by FRAP experiments that Sls does not exchange during a short time period.
This work is of significant value to a large audience. It provides a clear and precise pipeline for the generation of efficient nanobodies, which are invaluable tools of modern biology.
-
eLife assessment
The work describes the generation of novel reagents, nanobodies, which are single molecule antibodies from alpacas, which the authors raised against specific domains of two giant fly muscle proteins called Sallimus and Projectin. These nanobodies, combined with the so-called DNA-Paint approach, enabled the authors to reach an unprecedented spatial resolution and define the position of those domains. Thereby, the authors could propose a model for the organization and extent of those proteins along muscle sarcomeres.
-
Reviewer #1 (Public Review):
The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce the organization of …
Reviewer #1 (Public Review):
The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce the organization of the epitopes in different muscle types and propose a model for Sallimus and Projectin arrangement in muscles, including in larvae which are difficult to label with traditional antibodies due to their impermeable chitin skeleton. Finally, they could fuse the gene encoding one of the nanobodies to the open reading frame of NeonGreen and express the corresponding fusion protein in animals to use the probe in FRAP assays.
The work is very well performed and convincing. However, given its significant redundancy in terms of biological conclusions with the companion study "Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles" by the same authors, and other published papers, I recommend the authors further prove the use of their nanobodies in live assays. In particular, the authors should test whether they can use the nanobodies to induce protein degradation either permanently or conditionally.
-
Reviewer #2 (Public Review):
The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectin-specific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of …
Reviewer #2 (Public Review):
The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectin-specific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of homology of Drosophila Sallimus and Projectin with related proteins in other species is not discussed. Whether the nanobodies reported here would be useful in other (closely related?) species, therefore, remains to be established. For those studying muscle biology in Drosophila, the nanobodies described here will be publicly available as cDNAs. Ease of production implies a readily shared and standardized resource for the field.
Further characterization of these nanobodies by biochemical methods such as immunoblotting would be challenging, given the size of the target proteins. In view of the technical nature of this manuscript, the authors should perhaps critically discuss the distinction between bulky GFP tags versus the much smaller epitope tags and the nanobodies that recognize them, although this was covered in a recent eLife paper from the Perrimon lab. Insertion of small tags, in conjunction with nanobodies that recognize them, would be less perturbing than the much bulkier GFP tag and lend itself to genome-wide applications. Creating nanobodies uniquely specific for each protein encoded in the Drosophila genome is not realistic, and the targeted approach deployed here is obviously valuable.
The authors apply two different approaches to characterize the newly generated Nanobodies: more or less conventional immunohistochemistry with fluorescently labeled nanobodies, and in vivo expression of nanobodies fused to the fluorescent neongreen protein. The superiority of nanobodies in terms of tissue penetration has been shown by others in a direct comparison of intact fluorescently labeled immunoglobulins versus nanobodies. The authors state that in vivo labeling with nanobody fusions "thus far was done only with nanobodies against GFP, mCherry or short epitope tags." There is no fundamental difference between these recognition events and what the authors report for their Sallimus and Projectin-specific reagents. The section that starts at line 304 is thus a little bit of a 'straw man'. There is no reason to assume that a nanobody that recognizes a muscle protein would behave differently than a nanobody that would recognize that same protein (or another) when epitope- or GFP-tagged. What might be interesting is to examine the behavior of these muscle-specific nanobodies in the course of muscle contraction/relaxation: are there conformational alterations that promote dissociation of bound nanobodies? Do different nanobodies display discrete behavior in this regard? The manuscript is silent on how muscles behave in live L3 larvae. The FRAP experiment seems to suggest that not much is happening, but the text refers to the contraction of larval sarcomeres from 8.5 µM to 4.5 µM. Does the in vivo expressed nanobody remain stably bound during this contraction/relaxation cycle? What about the other nanobodies reported in this manuscript? Since the larval motion was reduced by exposure to diethylether, have the authors considered imaging the contractive cycle in the absence of such exposure?
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Reviewer #3 (Public Review):
Loreau et al. have presented a well-written manuscript reporting clever, original work taking advantage of fairly new biotechnology - the generation and use of single chain antibodies called nanobodies. The authors demonstrate the production of multiple nanobodies to two titin homologs in Drosophila and use these nanobodies to localize these proteins in several fly muscle types and discover interesting aspects of the localization and span of these elongated proteins in the muscle sarcomere. They also demonstrate that one of these single chain antibodies can be expressed in muscle fused to a fluorescent protein to image the localization of a segment of one of these giant proteins called Sallimus in muscle in a live fly. Their project is well-justified given the limitations of the usual approaches for …
Reviewer #3 (Public Review):
Loreau et al. have presented a well-written manuscript reporting clever, original work taking advantage of fairly new biotechnology - the generation and use of single chain antibodies called nanobodies. The authors demonstrate the production of multiple nanobodies to two titin homologs in Drosophila and use these nanobodies to localize these proteins in several fly muscle types and discover interesting aspects of the localization and span of these elongated proteins in the muscle sarcomere. They also demonstrate that one of these single chain antibodies can be expressed in muscle fused to a fluorescent protein to image the localization of a segment of one of these giant proteins called Sallimus in muscle in a live fly. Their project is well-justified given the limitations of the usual approaches for localizing and studying the dynamics of proteins in the muscle of model organisms such as the possibility that GFP tagging of a protein will interfere with its localization or function, and poor penetration of large IgG or IgM antibodies into densly packed structures like the sarcomere after fixation as compared to smaller nanbodies.
They achieved their goals consistent with the known/expected properties of nanobodies: (1) They demonstrate that at least one of their nanobodies binds with very high affinity. (2) They bind with high specificity. (3) The nanobodies show much better penetration of fixed stage 17 embryos than do conventional antibodies.
They use their nanobodies mostly generated to the N- and C-terminal ends of Sallimus and Projectin to learn new information about how these elongated proteins span and are oriented in the sarcomere. For example, in examining larval muscles which have long sarcomeres (8.5 microns), using nanobodies to domains located near the N- and C-termini, they show definitively that the predicted 2.1 MDa protein Sallimus spans the entire I-band and extends a bit into the A-band with its N-terminus embedded in the Z-disk and C-terminus in the outer edge of the A-band. Using a similar approach they also show that the 800 kDa Projectin decorates the entire myosin thick filament except for the H-zone and M-line in a polar orientation. Their final experiment is most exciting! They were able to express in fly larval muscles a nanobody directed to near the N-terminus of Sallimus fused to NeonGreen and show that it localizes to Z-disks in living larvae, and by FRAP experiments demonstrate that the binding of this nanobody to Sallimus in vivo is very stable. This opens the door to using a similar approach to study the assembly, dynamics, and even conformational changes of a protein in a complex in a live animal in real time.
There are only a few minor weaknesses about their conclusions: (1) They should note that in fact their estimate of the span of Sallimus could be an underestimate since their Nano2 nanobody is directed to Ig13/14 so if all of these 12 Ig domains N-terminal of their epitope were unwound it would add 12 X 30 nm = 360 nm of length, and even if unwound would add about 50 nm of length. (2) They discuss how Sallimus and Projectin are the two Drosophila homologs of mammalian titin, however, they ignore the fact that there is more similarity between Sallimus and Projectin to muscle proteins in invertebrates. For example, in C. elegans, TTN-1 is the counterpart of Sallimus, and twitchin is the counterpart of Projectin, both in size and domain organization. The authors present definitive data to support Figure 9, their nice model for a fly larval sarcomere but fail to point out that this model likely pertains to C. elegans and other invertebrates. In Forbes et al. (2010) it was shown that TTN-1, which can be detected by western blot as ~2 MDa protein and using two polyclonal antibodies spans the entire I-band and extends into the outer edge of the A-band, very similar to what the authors here have shown, more elegantly for Sallimus. In addition, several studies have shown that twitchin (Projectin) does not extend into the M-line; the M-line is exclusively occupied by UNC-89, the homolog of Obscurin.
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