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 H, Hatch V, Ali L, Lehman W, Craig RW, Tobacman LS. (2005). Mini-thin filaments regulated by troponin-tropomyosin. Craig Lab Publications. https://doi.org/10.1073/pnas.0407225102. Retrieved from https://escholarship.umassmed.edu/craig/18