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Analysis of molecular forces transmitted by Talin during muscle development in vivo
Analysis of molecular forces transmitted by Talin during muscle development in vivo
The muscle-tendon system built during the development of an animal is essential to allow the body to move, breath or keep the heart beating for a lifetime. The muscle is the most important force producing tissue in an animal and, at the same time, it is also dependent on forces built up in the muscle-tendon tissue, especially during its development. Using the Drosophila musculature as a model system, it had been shown that tension is built up in the muscle-tendon tissue during development and that this tension is required for myofibrillogenesis, the process of building myofibrils, which are long chains of the contractile units of muscles called sarcomeres. The main focus of this thesis was to analyze how tension in tissues is transmitted across proteins at the molecular level to understand how proteins sense and respond to mechanical forces in vivo. As a model system, the developing Drosophila flight muscles were used that form in the pupal stage of the Drosophila life cycle. During development, these muscles attach to tendon cells and the connections between these two cells, called muscle attachment sites, need to bear the forces built up in the tissue. Muscle attachments are cell-extracellular matrix (ECM)-cell contacts that require receptor molecules in the cell membrane called integrins to connect the ECM between the cells with the contractile actin cytoskeleton inside the cells. Since integrins cannot directly connect to actin themselves, they require an adaptor protein called Talin that can bind to both integrin and actin filaments. Thus, Talin is in the ideal position to transmit and sense forces at muscle attachments. Previous studies on Talin force transduction demonstrated that Talin indeed bears forces in the piconewton (pN) range using Förster resonance energy transfer (FRET)-based molecular tension sensors. However, these studies were based on analyzing Talin in focal adhesions in cells cultured in vitro in an artificial environment. Therefore, we aimed to analyze Talin force transmission for the first time in vivo in the natural mechanical environment in the intact organism. In a first step, different FRET-based tension sensor modules and various control constructs were inserted in Drosophila into the endogenous talin (rhea) gene, taking advantage of the newly established clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system to achieve precise modification of the genome. After demonstrating that the Talin protein is still fully functional after insertion of the tension sensor modules, forces across Talin were first quantified—as a proof of concept—in primary muscle fibers in vitro using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In a second step, forces transmitted by Talin at muscle attachments during flight muscle development were analyzed in detail in living pupae. We discovered that a surprisingly small proportion of Talin molecules at developing muscle attachments transmit forces at the same time (Paper I). Nevertheless, a large pool of Talin molecules need to be recruited to muscle attachment sites during development, as quantified by fluorescence correlation spectroscopy (FCS), to prepare for the forces generated by active muscle contractions in the adult fly. If the accumulation of Talin at flight muscle attachments is reduced during development by RNA interference (RNAi), the muscle attachments rupture in young adults, likely during the first flight attempts. In conclusion, recruitment of a high number of Talin molecules during development is physiologically relevant to enable the muscle to adapt to sudden changes in tissue forces, likely by dynamically sharing the load among the Talin molecules. This mechanical adaptation concept is important to ensure that the muscle-tendon connections are stable and last for a lifetime. During the course of the thesis, I also discovered that flight muscles contract spontaneously during development. Characterization of these contractions in wild-type animals compared to a knockdown condition provided a functional readout for myofibrillogenesis during development (Paper IV). Furthermore, a review article on the role of mechanical forces during muscle development (Paper II) and a video article explaining how to perform in vivo imaging in Drosophila pupae (Paper III) were published.
Talin, muscle attachment, Drosophila, integrin, tension sensor
Lemke, Sandra Beatrice
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Lemke, Sandra Beatrice (2019): Analysis of molecular forces transmitted by Talin during muscle development in vivo. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The muscle-tendon system built during the development of an animal is essential to allow the body to move, breath or keep the heart beating for a lifetime. The muscle is the most important force producing tissue in an animal and, at the same time, it is also dependent on forces built up in the muscle-tendon tissue, especially during its development. Using the Drosophila musculature as a model system, it had been shown that tension is built up in the muscle-tendon tissue during development and that this tension is required for myofibrillogenesis, the process of building myofibrils, which are long chains of the contractile units of muscles called sarcomeres. The main focus of this thesis was to analyze how tension in tissues is transmitted across proteins at the molecular level to understand how proteins sense and respond to mechanical forces in vivo. As a model system, the developing Drosophila flight muscles were used that form in the pupal stage of the Drosophila life cycle. During development, these muscles attach to tendon cells and the connections between these two cells, called muscle attachment sites, need to bear the forces built up in the tissue. Muscle attachments are cell-extracellular matrix (ECM)-cell contacts that require receptor molecules in the cell membrane called integrins to connect the ECM between the cells with the contractile actin cytoskeleton inside the cells. Since integrins cannot directly connect to actin themselves, they require an adaptor protein called Talin that can bind to both integrin and actin filaments. Thus, Talin is in the ideal position to transmit and sense forces at muscle attachments. Previous studies on Talin force transduction demonstrated that Talin indeed bears forces in the piconewton (pN) range using Förster resonance energy transfer (FRET)-based molecular tension sensors. However, these studies were based on analyzing Talin in focal adhesions in cells cultured in vitro in an artificial environment. Therefore, we aimed to analyze Talin force transmission for the first time in vivo in the natural mechanical environment in the intact organism. In a first step, different FRET-based tension sensor modules and various control constructs were inserted in Drosophila into the endogenous talin (rhea) gene, taking advantage of the newly established clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system to achieve precise modification of the genome. After demonstrating that the Talin protein is still fully functional after insertion of the tension sensor modules, forces across Talin were first quantified—as a proof of concept—in primary muscle fibers in vitro using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In a second step, forces transmitted by Talin at muscle attachments during flight muscle development were analyzed in detail in living pupae. We discovered that a surprisingly small proportion of Talin molecules at developing muscle attachments transmit forces at the same time (Paper I). Nevertheless, a large pool of Talin molecules need to be recruited to muscle attachment sites during development, as quantified by fluorescence correlation spectroscopy (FCS), to prepare for the forces generated by active muscle contractions in the adult fly. If the accumulation of Talin at flight muscle attachments is reduced during development by RNA interference (RNAi), the muscle attachments rupture in young adults, likely during the first flight attempts. In conclusion, recruitment of a high number of Talin molecules during development is physiologically relevant to enable the muscle to adapt to sudden changes in tissue forces, likely by dynamically sharing the load among the Talin molecules. This mechanical adaptation concept is important to ensure that the muscle-tendon connections are stable and last for a lifetime. During the course of the thesis, I also discovered that flight muscles contract spontaneously during development. Characterization of these contractions in wild-type animals compared to a knockdown condition provided a functional readout for myofibrillogenesis during development (Paper IV). Furthermore, a review article on the role of mechanical forces during muscle development (Paper II) and a video article explaining how to perform in vivo imaging in Drosophila pupae (Paper III) were published.