We will solve the structure of the sarcomere at nearatomic resolution, unravel the fundamentals of its force-driven assembly and turnover in health and ageing, and develop the foundations for future basic and translational research including the design and development of new agents to mitigate muscle disease and ageing.
Conserved Sarcomere architecture
Sarcomeres are small repeating subunits of myofibrils, the long cylinders that bundle together to make the muscle fibres. Inside the sarcomeres, filaments of the proteins myosin and actin interact to generate muscle contraction and relaxation.
While many sub-components are structurally and functionally characterised, muscle is more than the sum of these parts: its function is highly cooperative and its structure is dynamic over time and space. A precise molecular understanding of how the entire sarcomere machine forms and functions is required to understand its role in health, disease and ageing.
So far, traditional experimental approaches to investigate the structure and function of muscle tissue were performed on reconstructed protein complexes or suffered from low resolution.
This consortium will deploy an unparalleled complementary knowledge and technology base to address these fundamental and translational questions. We will solve the structure of the sarcomere at nearatomic resolution, unravel the fundamentals of its force-driven assembly and turnover in health and ageing, and develop the foundations for future basic and translational research including the design and development of new agents to mitigate muscle disease and ageing.
"Our muscles, both skeletal and heart muscles, have to function flawlessly for an entire life duration and must therefore be regularly serviced. We do not yet know how this works exactly. That is why we want to investigate and compare the composition and the structure of sarcomeres in young and aged muscles,“
Objective 1: Determine the nanostructure of the sarcomeres
The sarcomeric architecture and its molecular components are largely conserved from insects to mammals however, different muscle types display important functional differences. Thus, we will compare the sarcomere nanostructure from Drosophila flight muscle with fast skeletal muscles from zebrafish and mouse. These data will not only provide detailed information on the general evolutionarily conserved structural design
of sarcomeres, but also reveal important differences that explain functional specialization.
In order to determine the sarcomeric nanostructure of these three animal models in situ, we plan to extend our previous single particle cryo-EM work to cryo-FIB/cryo-ET.
Objective 2: Sarcomere assembly during muscle development in vivo
In our previous work, we discovered that Drosophila myofibrils self-organize in a tension-dependent manner and identified three distinct myofibrillogenesis phases by light microscopy. First, immature sarcomeres assemble into long myofibrils; second, additional immature sarcomeres are added to each myofibril to enable muscle fiber growth; third, all sarcomeres mature to pseudo-crystalline regularity. Similar simultaneous myofibril assembly stages also occur in zebrafish muscles, however, it is not known if myofibrillogenesis in fish follows the same tension-driven self-organization principle.
In order to understand the molecular mechanism and dynamics of sarcomere assembly and maturation, we plan to expand our studies to super-resolution light microscopy, cryo-ET and live imaging.
Objective 3: Sarcomere structure and maintenance during muscle aging
We hypothesize that the sarcomeric nanostructure is directly affected at old age due to the accumulation of damaged and abnormally modified proteins.
In order to understand at the molecular level if and how sarcomeres change their composition and structure during aging, we plan to isolate myofibrils from Drosophila and mice at different ages. To directly correlate the amount of mechanical force produced by a muscle with changes in sarcomeric structure and composition, we plan to generate muscles of defined exercise history from Drosophila and mice.
We will analyze the muscles by quantitative proteomics, measure sarcomeric protein turnover using pulsed feeding of heavy lysine (SILAC) and determine the nanostructure of their sarcomeres by super-resolution light microscopy and cryo-ET.