A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Jaime Andrés Rivas-Pardo, Yong Li, Zsolt Mártonfalvi, Rafael Tapia-Rojo, Andreas Unger, Ángel Fernández-Trasancos, Elías Herrero-Galán, Diana Velázquez-Carreras, Julio M. Fernández, Wolfgang A. Linke () and Jorge Alegre-Cebollada ()
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Jaime Andrés Rivas-Pardo: Columbia University
Yong Li: University of Muenster
Zsolt Mártonfalvi: Semmelweis University
Rafael Tapia-Rojo: Columbia University
Andreas Unger: University of Muenster
Ángel Fernández-Trasancos: Centro Nacional de Investigaciones Cardiovasculares (CNIC)
Elías Herrero-Galán: Centro Nacional de Investigaciones Cardiovasculares (CNIC)
Diana Velázquez-Carreras: Centro Nacional de Investigaciones Cardiovasculares (CNIC)
Julio M. Fernández: Columbia University
Wolfgang A. Linke: University of Muenster
Jorge Alegre-Cebollada: Centro Nacional de Investigaciones Cardiovasculares (CNIC)

Nature Communications, 2020, vol. 11, issue 1, 1-13

Abstract: Abstract Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces

Date: 2020
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DOI: 10.1038/s41467-020-15465-9

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