Elastic properties of single titin molecules made visible through fluorescent F-actin binding

Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle, driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti-titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titin - estimated by observing the breakage of the titin-bound actin filaments - may reach above ~ 100 pN (longitudinal tensile strength of actin. Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titin's molecular properties.