ELASTIC PROPERTIES OF SINGLE TITIN MOLECULES MADE VISIBLE THROUGH FLUORESCENT F-ACTIN BINDING

Titin (also known as connection) is a giant filamentous protein that s pans the distance between the Z- and M-lines of the vertebrate muscle sarcomere [1-4]. Several indirect observations have implicated titin a s playing a fundamental role in the generation of passive force of mus cle [5,6], driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstra ted. Here we have used the recently shown strong actin-binding propert y of titin [7-9] to indirectly visualize and manipulate single molecul es of titin. Titin molecules were immobilized on a microscope coversli p by attaching them to anti-titin antibody. The titin molecules were d etected by attaching fluorescent actin filaments to them. The titin mo lecules were subsequently stretched by manipulating the free end of th e attached actin filaments with a glass microneedle. Titin is shown he re to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followe d by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (ii ms time constant) was followed by a slowe r one (400 ms time constant). The force necessary to fully extend titi n-estimated by observing the breakage of the titin-bound actin filamen ts-may reach above similar to 100 pN (longitudinal tensile strength of actin [10]). Attachment of fluorescent actin filaments to titin provi des a useful tool to further probe titin's molecular properties. (C) 1 996 Academic Press, Inc.