A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Abstract

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 μm 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.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles

“…Nanobodies are only 13 kDa or less than 4 nm in size (Helma et al, 2015; Pleiner et al, 2015) and hence are ideal labels for two main reasons: their small size is first placing the label very close to the domain of interest and second allows efficient penetration into dense structures present in cells or protein complexes which enables high labelling density. This was demonstrated by the superior labelling abilities of our nanobodies compared to antibodies in stage 17 embryos in an accompanying manuscript (Loreau et al, 2022). As an attempt to verify if this is also the case in the crowded environment of mature flight muscles, we stained flight muscles with Sls-Nano2 (binding Sls-Ig13/14) and compared them to the endogenously expressed M-band protein Obscurin-GFP, or to a staining with an anti-Sls antibody (anti- Kettin, binding Sls-Ig16).…”

“…In order to verify the expression and to determine the precise location of the different Sls domains in adult flight muscle sarcomeres, we selected three different regions in Sls, against which we recently generated nanobodies (Loreau et al, 2022): Sls-Ig13/14, Sls-Ig49/50 and Sls-Ig51/Fn2, the first being relatively close to the N-terminus, the other two being close to the C-terminus of the Sls flight muscle isoform (Figure 1 – figure supplement 1A). Similarly, we selected two regions in Proj close to its N-terminus (Proj-Ig5-8 and Proj-Fn1/2) and two regions close to its C-terminus (Proj-Ig27-Fn35 and Proj-kinase domain).…”

“…Similarly, we selected two regions in Proj close to its N-terminus (Proj-Ig5-8 and Proj-Fn1/2) and two regions close to its C-terminus (Proj-Ig27-Fn35 and Proj-kinase domain). The generation of these nanobodies against Sls and Proj domains as well as their specificity, tested in Drosophila embryonic and larval muscles, were documented in an accompanying manuscript (Loreau et al, 2022).…”

“…Striated muscle architecture is not restricted to vertebrates but conserved in insects and worms. However, in contrast to vertebrates, titin’s role in Drosophila and C. elegans appears to be split into two proteins, one containing the flexible I-band features and the other the stiff A-band features of titin (Loreau et al, 2022; Tskhovrebova and Trinick, 2003). Surprisingly, the sarcomere length in flies and worms is still stereotypic for the respective muscle fiber type.…”

“…Here, we chose the Drosophila indirect flight muscles to determine the precise location of the two Drosophila homologs Sallimus (Sls) and Projectin (Proj). We selected key domains at different locations within Sls and Proj, against which we raised specific nanobodies (Loreau et al, 2022). We applied single and dual-colour DNA-PAINT super- resolution microscopy to intact flight muscle specimens, which determined the precise architecture of Sls and Proj in the flight muscle sarcomere.…”