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Functional analyses of the PEVK domains of the Drosophila titin homologs Projectin and Sallimus and the Projectin Kinase domain, a genetic approach
Functional analyses of the PEVK domains of the Drosophila titin homologs Projectin and Sallimus and the Projectin Kinase domain, a genetic approach
Connecting filaments like titin are giant proteins that are core components of the sarcomere, the smallest contractile unit of muscle. They fulfill key roles in the formation, structure and functioning of the muscles through their structure and specific sub-sarcomeric localization. Connecting filaments typically contain immunoglobulin domains, fibronectin type three domains, spring-like PEVK domains and a kinase domain. This allows connecting filaments to serve as a scaffold extending from the Z-disc border to the central thick filament of the sarcomere, to play a role in signaling pathways and to contribute to sarcomere (and thus muscle) stiffness, a key mechanical property of the muscle. While the giant titin molecule is the single connecting filament in vertebrates, other organisms may have several homologs. Drosophila has two functional titin homologs: Projectin and Sallimus. While Projectin contains a kinase domain like titin, Sallimus does not. In the asynchronous fibrillar flight muscles (IFMs, which power flight) both Sallimus and Projectin have been reported to connect the thick filaments to the Z-disc. However, in the tubular (skeletal) muscles, Projectin has been reported to completely localize on the thick filaments, while Sallimus connects the Z-disc to the thick filaments. Titin is expressed in many different isoforms, where stiff muscles like the vertebrate cardiac muscle contain short titin isoforms with short PEVK domains, while the more compliant skeletal muscles contain much larger titin isoforms with large and thus compliant PEVK domains. Similarly in Drosophila, the very stiff IFMs contain short Sallimus and Projectin isoforms with very short or completely missing PEVK domains, while the tubular muscles contain much larger isoforms with larger PEVK domains. In addition, the parallel localization of Projectin and Sallimus could further increase the stiffness of IFMs. Despite the large body of work on titin, Projectin and Sallimus, there are still many open questions. Work on the soleus muscle in vertebrates, for example, suggests that titin may not always significantly contribute to muscle stiffness. In other animal groups like insects, there is actually very little known about muscle stiffness apart from the IFMs. Currently the actual importance of the Projectin and Sallimus related stiffness in the Drosophila tubular muscles is mostly extrapolated from data on vertebrate titin. In addition, the function and precise localization of the Projectin kinase domain remains to be demonstrated. This work aimed to experimentally assess the contribution of the PEVK domains of both Projectin and Sallimus and the Projectin kinase domain to the structural and functional properties of the Drosophila muscles in vivo through genetic approaches (BAC recombineering and CRISPR- Cas9). The lengths of the PEVK domains in both Projectin and Sallimus were drastically shortened. Using endogenous tags, I assessed the localization patterns of the Projectin PEVK domain and two of the very large PEVK exons in Sallimus in different muscle types. I was able to demonstrate that the Projectin PEVK domain and two large PEVK exons in Sallimus localize in the I-band region of the sarcomere (the region between a Z-disc and the thick filament) regardless of the muscle type. This supports the connection between the Z-disc and the thick filaments through Projectin in the IFMs. In the tubular muscles, the Projectin N-terminus enters the I-band from the thick filaments, but does not reach the Z-disc. The localization patterns of the studied PEVK exons of Sallimus matched previous descriptions and predictions in the literature. Surprisingly, strong reductions in the size of the PEVK domains of Projectin or Sallimus did not result in any obvious defects in the structure or functioning of the tubular muscles. This indicates that either the length of the PEVK domains does not play a significant role in the structural or functional properties of the tubular muscles or that there is a strong compensation capability.
Drosophila, titin, stiffness, muscle function and structure, CRISPR/Cas9, Projectin Sallimus, PEVK
Koolhaas, Wouter
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Koolhaas, Wouter (2018): Functional analyses of the PEVK domains of the Drosophila titin homologs Projectin and Sallimus and the Projectin Kinase domain, a genetic approach. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Connecting filaments like titin are giant proteins that are core components of the sarcomere, the smallest contractile unit of muscle. They fulfill key roles in the formation, structure and functioning of the muscles through their structure and specific sub-sarcomeric localization. Connecting filaments typically contain immunoglobulin domains, fibronectin type three domains, spring-like PEVK domains and a kinase domain. This allows connecting filaments to serve as a scaffold extending from the Z-disc border to the central thick filament of the sarcomere, to play a role in signaling pathways and to contribute to sarcomere (and thus muscle) stiffness, a key mechanical property of the muscle. While the giant titin molecule is the single connecting filament in vertebrates, other organisms may have several homologs. Drosophila has two functional titin homologs: Projectin and Sallimus. While Projectin contains a kinase domain like titin, Sallimus does not. In the asynchronous fibrillar flight muscles (IFMs, which power flight) both Sallimus and Projectin have been reported to connect the thick filaments to the Z-disc. However, in the tubular (skeletal) muscles, Projectin has been reported to completely localize on the thick filaments, while Sallimus connects the Z-disc to the thick filaments. Titin is expressed in many different isoforms, where stiff muscles like the vertebrate cardiac muscle contain short titin isoforms with short PEVK domains, while the more compliant skeletal muscles contain much larger titin isoforms with large and thus compliant PEVK domains. Similarly in Drosophila, the very stiff IFMs contain short Sallimus and Projectin isoforms with very short or completely missing PEVK domains, while the tubular muscles contain much larger isoforms with larger PEVK domains. In addition, the parallel localization of Projectin and Sallimus could further increase the stiffness of IFMs. Despite the large body of work on titin, Projectin and Sallimus, there are still many open questions. Work on the soleus muscle in vertebrates, for example, suggests that titin may not always significantly contribute to muscle stiffness. In other animal groups like insects, there is actually very little known about muscle stiffness apart from the IFMs. Currently the actual importance of the Projectin and Sallimus related stiffness in the Drosophila tubular muscles is mostly extrapolated from data on vertebrate titin. In addition, the function and precise localization of the Projectin kinase domain remains to be demonstrated. This work aimed to experimentally assess the contribution of the PEVK domains of both Projectin and Sallimus and the Projectin kinase domain to the structural and functional properties of the Drosophila muscles in vivo through genetic approaches (BAC recombineering and CRISPR- Cas9). The lengths of the PEVK domains in both Projectin and Sallimus were drastically shortened. Using endogenous tags, I assessed the localization patterns of the Projectin PEVK domain and two of the very large PEVK exons in Sallimus in different muscle types. I was able to demonstrate that the Projectin PEVK domain and two large PEVK exons in Sallimus localize in the I-band region of the sarcomere (the region between a Z-disc and the thick filament) regardless of the muscle type. This supports the connection between the Z-disc and the thick filaments through Projectin in the IFMs. In the tubular muscles, the Projectin N-terminus enters the I-band from the thick filaments, but does not reach the Z-disc. The localization patterns of the studied PEVK exons of Sallimus matched previous descriptions and predictions in the literature. Surprisingly, strong reductions in the size of the PEVK domains of Projectin or Sallimus did not result in any obvious defects in the structure or functioning of the tubular muscles. This indicates that either the length of the PEVK domains does not play a significant role in the structural or functional properties of the tubular muscles or that there is a strong compensation capability.