The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of <i>Drosophila</i>

Katzemich, Anja; Kreisköther, Nina; Alexandrovich, Alexander; Elliott, Christopher J.H.; Schöck, Frieder; Leonard, Kevin; Sparrow, J M; Bullard, Belinda

doi:10.1242/jcs.119552

Cited by 18 publications

(12 citation statements)

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“…We wanted to verify our surprising finding that N- and C We hypothesized that the small central gap visible in the Proj-kinase domain nanobody patterns is caused by a Projectin-free M-band of the larval sarcomere. Co-labelling the M-band with Obscurin-GFP, which specifically localises to the M-band (Katzemich et al, 2012;Sarov et al, 2016), confirmed that the gap present in the Proj-kinase nanobody pattern is consistent with its absence from the M-band (Figure 7D). Taken together, our results demonstrate that Projectin decorates the myosin filament in a defined polar orientation, likely from the tip of the myosin filament until the beginning of the H-zone that is devoid of myosin heads.…”

Section: Projectin Is Oriented On the Thick Filamentsupporting

confidence: 61%

“…As muscle and in particular sarcomere architecture is well-conserved, Drosophila is a fantastic model to study how a sarcomere is built during development (Katzemich et al, 2013; 2015; Orfanos et al, 2015; Weitkunat et al, 2017; 2014). Generation of monoclonal antibodies against Drosophila sarcomere proteins have been insightful to locate key proteins within the mature sarcomere (Burkart et al, 2007; Ferguson et al, 1994; Katzemich et al, 2012; Lakey et al, 1990; Qiu et al, 2005; Szikora et al, 2020). However, a systematic toolbox of antibodies recognising defined domains of the often large sarcomeric proteins, in particular against defined domains of the two large Drosophila titin homologs Sallimus (Sls) and Projectin (gene called bent, bt ) is still missing.…”

Section: Introductionmentioning

confidence: 99%

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A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Schnorrer

Loreau²,

Rees

et al. 2022

Preprint

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Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two large Drosophila titin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 μm to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal the in vivo dynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

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Section: Projectin Is Oriented On the Thick Filamentsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Schnorrer

Loreau²,

Rees

et al. 2022

Preprint

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“…Reduced expression of Unc ‐ 89 using P‐element insertion results in flightless adults in D . melanogaster (Katzemich et al, 2012). Upregulation of these genes in Y M males at lower temperatures might improve muscle performance.…”

Section: Discussionmentioning

confidence: 99%

Temperature‐dependent effects of house fly proto‐Y chromosomes on gene expression could be responsible for fitness differences that maintain polygenic sex determination

et al. 2021

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Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto‐sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto‐sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female‐determiner on one of the chromosomes as well. The two most common male‐determining proto‐Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature‐dependent fitness effects could be manifested through temperature‐dependent gene expression differences across proto‐Y chromosome genotypes. These gene expression differences may be the result of cis regulatory variants that affect the expression of genes on the proto‐sex chromosomes, or trans effects of the proto‐Y chromosomes on genes elswhere in the genome. We used RNA‐seq to identify genes whose expression depends on proto‐Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature‐dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time‐point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto‐Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype‐by‐temperature interactions on expression were not enriched on the proto‐sex chromosomes. Moreover, there was no evidence that temperature‐dependent expression is driven by chromosome‐wide cis‐regulatory divergence between the proto‐Y and proto‐X alleles. Therefore, if temperature‐dependent gene expression is responsible for differences in phenotypes and fitness of proto‐Y genotypes across house fly populations, these effects are driven by a small number of temperature‐dependent alleles on the proto‐Y chromosomes that may have trans effects on the expression of genes on other chromosomes.

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“…The cardiac isoform of myosin-binding protein C (MyBP-C), encoded by MYBPC3, binds to titin (encoded by TTN) and light meromyosin (LMM, the tail region of myosin) in the A-band via its C-terminal domain [8,9]. In the M-band, the myomesin family, namely, myomesin, M-protein/myomesin-2 and myomesin-3, encoded by MYOM1, MYOM2 and MYOM3, respectively in humans [10][11][12], acts as a structural sarcomere stabilizer by cross-connecting thick filaments [13][14][15].…”

Section: (A) Thick Filamentmentioning

confidence: 99%

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles

Ahmed

Tokuyama

Anzai

et al. 2022

Phil. Trans. R. Soc. B

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During postnatal cardiac development, cardiomyocytes mature and turn into adult ones. Hence, all cellular properties, including morphology, structure, physiology and metabolism, are changed. One of the most important aspects is the contractile apparatus, of which the minimum unit is known as a sarcomere. Sarcomere maturation is evident by enhanced sarcomere alignment, ultrastructural organization and myofibrillar isoform switching. Any maturation process failure may result in cardiomyopathy. Sarcomere function is intricately related to other organelles, and the growing evidence suggests reciprocal regulation of sarcomere and mitochondria on their maturation. Herein, we summarize the molecular mechanism that regulates sarcomere maturation and the interplay between sarcomere and other organelles in cardiomyocyte maturation. This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

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The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila

Cited by 18 publications

References 0 publications

A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Temperature‐dependent effects of house fly proto‐Y chromosomes on gene expression could be responsible for fitness differences that maintain polygenic sex determination

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles

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