Agenda

Skeletal muscles, responsible for animal movement, are packed with the repeating functional unit characteristic of striated muscles: the sarcomere. Sarcomeres are super-assemblies of long and ordered actin and myosin filaments, linked by titin, the largest human protein. Actin crosslinkers, including Zasp52 and ฆม-Actinin, are located at the Z-disc, bordering each sarcomere, whereas myosin crosslinkers, including Obscurin, are located at the M-band, in the sarcomere center. While the muscle contraction mechanism is well characterized, the ultrastructure of sarcomeres and, in particular, how the sarcomere is built during development remains poorly understood. During my thesis, I combined the super-resolution method DNA-PAINT with a library of newly developed nanobodies recognizing specific sarcomere protein domains to uncover the ultrastructural organisation of sarcomeres in mature (Aim 1) and developing (Aim 2) indirect flight muscles of Drosophila. To address Aim 1, I imaged muscle tissue at 5 nm resolution and first focused on the two Drosophila titin homologs Sallimus and Projectin. We found that Sallimus and Projectin are linearly arranged in the sarcomere, displaying a staggered organisation: Sallimus locates its N-terminus at the center of the Z-disc and spans across the entire 90 nm I-band, reaching the myosin filament with its C-terminus; Projectin covers the distal 260 nm of the myosin containing A-band, and its N-terminal region overlaps with the Sallimus C-terminal region over 10 nm at the distal end of the A-band. Furthermore, I found that Zasp52 and ฆม-Actinin concentrate in a 90 nm wide region centered on the Z-disc, which defines the cross-linked area of the actin filaments. This aligns well with the high Z-disc density observed previously with electron microscopy. It suggests that both Zasp52 and ฆม-Actinin are arranged in multiple rows, instead of 2 distinct bands as had been published before. Next, I characterized the orientation of Obscurin at the M-band: I found that Obscurin spans 60 nm with its C-terminus in the center of the M-band and the N-terminus facing towards the myosin motor domains, likely crosslinking neighboring myosin filaments. This defines the first oriented protein in the sarcomere M-band. Finally, I analyzed the distribution of myosin binding proteins in the A-band, which makes up the bulk of the sarcomere. In addition to my finding that Projectin points out from the distal end of the A-band into the I-band, I discovered that Stretchin, another A-band protein that provides mechanical integrity to the sarcomeres, occupies almost the entire myosin filament, except for the Obscurin and Projectin regions. Together, this defines the dimensions of the flight muscle sarcomere at unprecedented resolution. To investigate the function of these subdivisions, we knocked down Projectin to see if the Stretchin pattern changes. However, this is not the case, suggesting a difference in A-band structure and not Projectin per se that prevents Stretchin from decorating the entire thick filament. During Aim 2, I set out to better understand sarcomere morphogenesis during development. Thus, I performed DNA-PAINT experiments using developmental flight muscles from pupae 48, 60, and 72 h after puparium formation (APF). Interestingly, I found that the initial Z-disc assemblies formed at 48 h APF are broader than 90 nm, and the initial I-band is longer than in the adult sarcomere. This suggests that the Z-disc compacts during sarcomere maturation, possibly by better ordering the protein components. Notably, during this phase, the force across the I-band titin Sallimus reduces or its isoform composition changes, as a shorter Sls isoform is expressed during flight muscle sarcomere maturation. At the same time, the length of the actin and myosin filaments and their overlap continuously increase. Together, DNA-PAINT super-resolution enabled us to better define the sarcomere dimensions in mature and developing flight muscles.