A nanobody toolbox to investigate localisation and dynamics of Drosophila titins - Archive ouverte HAL
Pré-Publication, Document De Travail Année : 2022

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins

Renate Rees
  • Fonction : Auteur
Waltraud Taxer
  • Fonction : Auteur
Kathrin Gregor
  • Fonction : Auteur
Bianka Mußil
  • Fonction : Auteur
Christophe Pitaval
  • Fonction : Auteur
Pierre Mangeol
Dirk Görlich
  • Fonction : Auteur

Résumé

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.

Dates et versions

hal-03798628 , version 1 (05-10-2022)

Identifiants

Citer

Vincent Loreau, Renate Rees, Eunice Hoyee Chan, Waltraud Taxer, Kathrin Gregor, et al.. A nanobody toolbox to investigate localisation and dynamics of Drosophila titins. 2022. ⟨hal-03798628⟩
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