Linke, W. A. | bioRxiv Science

bioRxiv2020

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

cell biology↗

bioRxiv2019

A HaloTag-TEV genetic cassette for mechanical phenotyping of native proteins

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 native molecules in their natural environment remains inaccessible to conventional genetics, biophysics and molecular biology tools. To address these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titins intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of titin under 1-100 pN forces using recently developed magnetic tweezers. At pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors. Insertion of the HaloTag-TEV cassette in proteins with mechanical roles opens new grounds to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

biophysics↗