Pitaval, C. | Search bioRxiv

bioRxiv2024

Titin-dependent biomechanical feedback tailors sarcomeres to specialised muscle functions in insects

Sarcomeres are the contractile units of muscles that enable animals to move. Insect muscles are remarkable examples because they use extremely different contraction frequencies (ranging from [~]1 to 1000 Hz) and amplitudes for flying, walking and crawling. This is puzzling because sarcomeres are built from essentially the same actin-myosin components. We show here that the giant protein titin is the key to this functional specialisation. I-band titin spans and determines the length of the sarcomeric I-band, and occurs in muscle-type-specific isoforms. Surprisingly, it also rules the length of the force-generating myosin filament in a force feedback mechanism, even though it is not present there. We provide evidence for this model and its validity beyond insects. SummaryHere we identified a mechanical mechanism that instructs sarcomeres to fulfill the specific needs of different muscle types.

developmental biology↗

bioRxiv2022

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two large Drosophila titin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 {micro}m to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal the in vivo dynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

developmental biology↗