Periodic Reporting for period 2 - StuDySARCOMERE (Structure and Cellular Dynamics of the Sarcomere)
Berichtszeitraum: 2021-08-01 bis 2023-01-31
Muscles are specialized tissues that power voluntary, fast and coordinated movements, a hallmark of animal life. The force-generating and load-bearing devices of muscles are called sarcomeres, highly structured ensembles of many proteins. 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 dynamic over time and space. The high-resolution structure of sarcomeres is unknown, yet a precise molecular understanding of how the entire sarcomere machine forms and functions is required to understand its role in health, disease and ageing. 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 near-atomic 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.
The consortium made significant progress on all three objectives of the grant. The Consortium determined the molecular architecture of native vertebrate skeletal sarcomeres, revealing three-dimensional sarcomere organization and the mechanism of thin filament regulation through nebulin. A combination of specific nanobody-labeling, time-lapse microscopy, and super-resolution microscopy enabled the consortium to investigate the dynamics of sarcomere formation and maintenance in different model muscles from different organisms.
Applying our Drosophila nanobody toolbox to study Drosophila titin homologs (Loreau et al. 2022, eLife), we revealed a staggered organisation of the two Drosophila titin homologs and found that one of the Drosophila titins extends over a length of 2.5 µm in larval sarcomeres (Schueder et al. 2022, eLife). This is significant as before our work the longest natively stretched sarcomeric protein was considered to be human titin, with a length of < 2 µm.
For the first time, our cryo-ET structures of mouse psoas muscle revealed the three-dimensional organization of the sarcomere, the different organization of myosin double heads and uncovered that alpha-actinin forms an irregular mesh of double cross-links between antiparallel actin filaments (Wang, Grange et al. 2021, Cell). Our recent cryo-ET structure of the thin filament from skeletal mouse muscle (Wang, Grange et al. 2022, Science) represents the highest resolution structure of a filament determined in situ and the only pseudo-atomic structure determined from tissues after cryo focused-ion-laser beam milling to date. Fundamentally, this study represents a leap in capability for structural biology and the ability to perform atomic-level structural analyses within native tissue will be at the forefront of molecular diagnostics in the years to come. In general our results highlight the strength of our synergistic approach integrating information from fly, fish and mammalian sarcomeres. In the next funding period we expect to obtain structures of the thick filament in relaxed and contracted states and of other sarcomeric domains from Drosophila, zebrafish and mouse. In addition, we plan to put our focus on objective 3, namely the investigation of the sarcomere structure and maintenance during muscle ageing.
For the first time, our cryo-ET structures of mouse psoas muscle revealed the three-dimensional organization of the sarcomere, the different organization of myosin double heads and uncovered that alpha-actinin forms an irregular mesh of double cross-links between antiparallel actin filaments (Wang, Grange et al. 2021, Cell). Our recent cryo-ET structure of the thin filament from skeletal mouse muscle (Wang, Grange et al. 2022, Science) represents the highest resolution structure of a filament determined in situ and the only pseudo-atomic structure determined from tissues after cryo focused-ion-laser beam milling to date. Fundamentally, this study represents a leap in capability for structural biology and the ability to perform atomic-level structural analyses within native tissue will be at the forefront of molecular diagnostics in the years to come. In general our results highlight the strength of our synergistic approach integrating information from fly, fish and mammalian sarcomeres. In the next funding period we expect to obtain structures of the thick filament in relaxed and contracted states and of other sarcomeric domains from Drosophila, zebrafish and mouse. In addition, we plan to put our focus on objective 3, namely the investigation of the sarcomere structure and maintenance during muscle ageing.