Title

Mini-thin filaments regulated by troponin-tropomyosin

UMMS Affiliation

Department of Cell Biology

Date

1-13-2005

Document Type

Article

Medical Subject Headings

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

Disciplines

Cell Biology

Abstract

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.

Rights and Permissions

Citation: 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

Related Resources

Link to Article in PubMed