Zasp52’s differentially expressed intrinsically disordered region confers thin filament stability at the Z-disc

Reviewer #1 (Public review):

The manuscript by Ho and Schock investigates the role of the Z-disc protein Zasp52 during Drosophila flight muscle development. It was known before, mainly by findings from this group, that Zasp52 is required for normal sarcomere morphogenesis, specifically Z-disc morphogenesis in indirect flight muscles. But the exact molecular mechanism by which Zasp52 contributes, apart from the fact that it is localised there and is somehow involved in multimerization/cross-linking, was not clear. This paper proposes that an intrinsically disordered region (IDR) in Zasp52 is needed for some of its functions, by stabilising Zasp52 localisation at the Z-disc. Specifically, the IDR in Zasp52 is proposed to be required for Z-disc maintenance during the mechanical challenges of flight, while being dispensable for the initial morphogenesis during development. This hypothesis is supported by strong genetic evidence and behavioural tests, deleting Zasp's IDR impairs flight from mid-age onwards, while a block in flight activity lifts the phenotype.

However, some of the phenotypic analysis, in particular the bending of the sarcomere, likely upon mechanical challenge by muscle contractions, needs more detailed investigations to be fully convincing.

Strengths:

(1) The linker in the alternatively spliced exon 15 of Zasp52 was deleted with a state-of-the-art genetic editing strategy. Surprisingly, flies are homozygous viable, showing that this long part of the Zasp52 protein is not essential for animal survival or sarcomere morphogenesis.

(2) The observed sarcomere phenotypes with age, especially the bending Z-discs, are new and exciting.

(3) The displayed EM images document interesting phenotypes.

(4) Most of the observed phenotypes can be rescued by re-expression of the long Zasp52 isoform, which does contain the IDR region, but not by a shorter one without it, suggesting that IDR is important.

(5) FRAP data measure the local turnover of a short-ZaspGFP and show that this increased in the Zasp mutant lacking the IDR domain, suggesting that Zasp-IDR might stabilise Zasp at the Z-disc.

(6) Interestingly, flight and sarcomere morphology phenotypes can be rescued by preventing the flies from flying, suggesting that they are mechanically induced.

Weaknesses:

(1) The western blot quantifications of Zasp isoform expression are weak. No error bars are indicated in the quantifications; the quantifications appear to be more qualitative than quantitative. According to band intensities, the long Zasp isoforms seem to be less present compared to the shorter ones, even in the flight muscles.

(2) The phenotypic analysis of the sarcomere appears somewhat superficial throughout the paper. Only Zasp52 and phalloidin are shown; no other Z-disc or thick filament proteins. At least myosin stainings and overview images are important to better judge the phenotypic variations. Are the variants between individuals or regional in the same muscle?

(3) EM images would benefit from better quantification.

(4) Other proteins were not analysed with the FRAP-based turnover assay for comparison in wild type and mutant. All Z-proteins might turn over faster in the mutant with the defective Z-disc.

Reviewer #2 (Public review):

Summary and Strengths:

This in-depth genetic analysis of Zasp52 function in Drosophila indirect flight muscle (IFM) provides an interesting perspective regarding the role of a partially disordered region (IDR) in exon 15e. This exon seems to be exclusively present in IFM and contributes to the prevention of myofibril disintegration during aging, likely due to interactions of this region with Z-disc insertion and/or stability. The addition of an isoform (PR) that lacks exon 15e serves as a nice control to illustrate the necessity of exon 15e in muscle structure and function. Overall, the manuscript is exceptionally well-written, logical, with nicely controlled experiments and detailed statistical analysis that largely support the conclusions drawn by the authors. While exon 15e is clearly involved in preventing muscle degeneration, a solid role for thin filament stability is not clearly shown (as mentioned in the abstract). In addition, which regions/how the proteins of the IDR may contribute are unclear.

Weaknesses:

(1) It is not clear in Figure S1A where exon 15e fits within the Zasp52 locus schematic. This is important as a premise of this paper describes this region to be key, and proof from multiple prediction programs would lend more weight to the prediction of the exon being largely disordered. Inclusion of the discussed short linear motifs, comparison with Canoe or LBD3 for similarities and/or an Alphafold structure would help make the authors' point (colorized with known domains).

(2) Interesting that immobilization rescues the deterioration phenotypes. The authors should explain in more detail how this was done to avoid dehydration/starvation of the flies.

(3) There is a lot of discussion about the potential function of the IDR region, specifically a putative actin binding motif or other 'ordered' regions that may contain short linear motifs. It would strengthen the findings to show which of these may be essential for Zasp52 function in the IFM. The ability to bind actin could be tested biochemically, and/or smaller deletions could be made to unequivocally test the role of the ABD vs other predicted motifs using genetics. If some of these regions are more ordered, where do they lie within, and do they form a predicted fold or structure that gives insight into function?

Author response:

Public Reviews:

Reviewer #1 (Public review):

The manuscript by Ho and Schock investigates the role of the Z-disc protein Zasp52 during Drosophila flight muscle development. It was known before, mainly by findings from this group, that Zasp52 is required for normal sarcomere morphogenesis, specifically Z-disc morphogenesis in indirect flight muscles. But the exact molecular mechanism by which Zasp52 contributes, apart from the fact that it is localised there and is somehow involved in multimerization/cross-linking, was not clear. This paper proposes that an intrinsically disordered region (IDR) in Zasp52 is needed for some of its functions, by stabilising Zasp52 localisation at the Z-disc. Specifically, the IDR in Zasp52 is proposed to be required for Z-disc maintenance during the mechanical challenges of flight, while being dispensable for the initial morphogenesis during development. This hypothesis is supported by strong genetic evidence and behavioural tests, deleting Zasp's IDR impairs flight from mid-age onwards, while a block in flight activity lifts the phenotype.

However, some of the phenotypic analysis, in particular the bending of the sarcomere, likely upon mechanical challenge by muscle contractions, needs more detailed investigations to be fully convincing.

Strengths:

(1) The linker in the alternatively spliced exon 15 of Zasp52 was deleted with a state-of-the-art genetic editing strategy. Surprisingly, flies are homozygous viable, showing that this long part of the Zasp52 protein is not essential for animal survival or sarcomere morphogenesis.

(2) The observed sarcomere phenotypes with age, especially the bending Z-discs, are new and exciting.

(3) The displayed EM images document interesting phenotypes.

(4) Most of the observed phenotypes can be rescued by re-expression of the long Zasp52 isoform, which does contain the IDR region, but not by a shorter one without it, suggesting that IDR is important.

(5) FRAP data measure the local turnover of a short-ZaspGFP and show that this increased in the Zasp mutant lacking the IDR domain, suggesting that Zasp-IDR might stabilise Zasp at the Z-disc.

(6) Interestingly, flight and sarcomere morphology phenotypes can be rescued by preventing the flies from flying, suggesting that they are mechanically induced.

Weaknesses:

(1) The western blot quantifications of Zasp isoform expression are weak. No error bars are indicated in the quantifications; the quantifications appear to be more qualitative than quantitative. According to band intensities, the long Zasp isoforms seem to be less present compared to the shorter ones, even in the flight muscles.

We will work on including quantifications with error bars for the Western blots in our resubmission. It is important to keep in mind that the main point in figure 1B is that there are plenty of exon15e-containing isoforms in IFM, in contrast to other tissues with very limited exon15e-containing isoforms. This is confirmed by the analysis of RNAseq data in figure 1C, and of course, by the flightless phenotype of the exon15e mutant.

(2) The phenotypic analysis of the sarcomere appears somewhat superficial throughout the paper. Only Zasp52 and phalloidin are shown; no other Z-disc or thick filament proteins. At least myosin stainings and overview images are important to better judge the phenotypic variations. Are the variants between individuals or regional in the same muscle?

Our images are representative of the observed phenotypes. We aim to provide overview images and other stainings to better illustrate the phenotypic variations in the revised version. Phenotypes are consistently present across all individuals, as reflected in our replicates. Interestingly, they appear to not be randomly interspersed among the sarcomeres but concentrated in certain regions of muscle more than others.

(3) EM images would benefit from better quantification.

We do not believe that EM images can be meaningfully quantified, because of the many selection steps preceding image acquisition.

(4) Other proteins were not analysed with the FRAP-based turnover assay for comparison in wild type and mutant. All Z-proteins might turn over faster in the mutant with the defective Z-disc.

This is the point we are trying to make. The Zasp52 IDR acts like a glue stabilizing all Z-disc proteins. We performed this experiment as a first step to explore whether an exon15e-lacking system exhibited modified dynamics, and we aim to provide more data in the revised version.

Reviewer #2 (Public review):

Summary and Strengths:

This in-depth genetic analysis of Zasp52 function in Drosophila indirect flight muscle (IFM) provides an interesting perspective regarding the role of a partially disordered region (IDR) in exon 15e. This exon seems to be exclusively present in IFM and contributes to the prevention of myofibril disintegration during aging, likely due to interactions of this region with Z-disc insertion and/or stability. The addition of an isoform (PR) that lacks exon 15e serves as a nice control to illustrate the necessity of exon 15e in muscle structure and function. Overall, the manuscript is exceptionally well-written, logical, with nicely controlled experiments and detailed statistical analysis that largely support the conclusions drawn by the authors. While exon 15e is clearly involved in preventing muscle degeneration, a solid role for thin filament stability is not clearly shown (as mentioned in the abstract). In addition, which regions/how the proteins of the IDR may contribute are unclear.

Weaknesses:

(1) It is not clear in Figure S1A where exon 15e fits within the Zasp52 locus schematic. This is important as a premise of this paper describes this region to be key, and proof from multiple prediction programs would lend more weight to the prediction of the exon being largely disordered. Inclusion of the discussed short linear motifs, comparison with Canoe or LBD3 for similarities and/or an Alphafold structure would help make the authors' point (colorized with known domains).

We will add a bar below figure S2A to show the region corresponding to exon 15e. We used three disorder prediction programs and one structure (order) prediction program. The majority of exon15e is completely disordered and of very low confidence score, and thus uninformative to display as an Alphafold structure. Likewise, IDR’s are very difficult to classify, therefore we cannot say much more than that LDB3, Zasp52, and Canoe contain IDRs, with Zasp52 and Canoe both having an actin-binding domain within the IDR. We will provide more data on the function of the ABD in the revised version.

(2) Interesting that immobilization rescues the deterioration phenotypes. The authors should explain in more detail how this was done to avoid dehydration/starvation of the flies.

We will provide more details in the revised version.

(3) There is a lot of discussion about the potential function of the IDR region, specifically a putative actin binding motif or other 'ordered' regions that may contain short linear motifs. It would strengthen the findings to show which of these may be essential for Zasp52 function in the IFM. The ability to bind actin could be tested biochemically, and/or smaller deletions could be made to unequivocally test the role of the ABD vs other predicted motifs using genetics. If some of these regions are more ordered, where do they lie within, and do they form a predicted fold or structure that gives insight into function?

We will provide data on the function of the ABD in the revised version.