Mini-thin filaments regulated by troponin-tropomyosin
Department of Cell Biology
Actins; Allosteric Regulation; Animals; Ca(2+) Mg(2+)-ATPase; Calcium; Cattle; Gelsolin; Macromolecular Substances; Microfilaments; Microscopy, Electron; Muscle, Skeletal; Myosin Subfragments; Particle Size; Protein Binding; Tropomyosin; Troponin
Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.
DOI of Published Version
Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):656-61. Epub 2005 Jan 11. Link to article on publisher's site
Proceedings of the National Academy of Sciences of the United States of America
Gong, Huiyu; Hatch, Victoria; Ali, Laith; Lehman, William; Craig, Roger W.; and Tobacman, Larry S., "Mini-thin filaments regulated by troponin-tropomyosin" (2005). Craig Lab Publications. 18.