| Autor: |
Vincent Loreau, Renate Rees, Eunice HoYee Chan, Waltraud Taxer, Kathrin Gregor, Bianka Mußil, Christophe Pitaval, Nuno Miguel Luis, Pierre Mangeol, Frank Schnorrer, Dirk Görlich |
| Přispěvatelé: |
Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS), Nanobiophysique, Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Popis: |
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 largeDrosophilatitin 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 thein vivodynamics 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 inDrosophilaor mammals. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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