Direct observation of tropomyosin binding to actin filaments

William M. Schmidt; William Lehman; Jeffrey R. Moore

doi:10.1002/cm.21225

journal article Jun 01, 2015

Direct observation of tropomyosin binding to actin filaments

William M. Schmidt William Lehman

Jeffrey R. Moore

Cytoskeleton Vol. 72 No. 6 pp. 292-303 · Wiley

View at Publisher Save 10.1002/cm.21225

Abstract

Tropomyosin is an elongated α‐helical coiled coil that binds to seven consecutive actin subunits along the long‐pitch helix of actin filaments. Once bound, tropomyosin polymerizes end‐to‐end and both stabilizes F‐actin and regulates access of various actin‐binding proteins including myosin in muscle and nonmuscle cells. Single tropomyosin molecules bind weakly to F‐actin with millimolar Kd, whereas the end‐to‐end linked tropomyosin associates with about a 1000‐fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In this study, we used total internal reflection fluorescence microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin‐stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites after random low‐affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on the concentration of tropomyosin, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F‐actin leads to discontinuous end‐to‐end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin‐bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. © 2015 Wiley Periodicals, Inc.

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Details

Published: Jun 01, 2015
Vol/Issue: 72(6)
Pages: 292-303
License: View

Authors

W

William M. Schmidt

Boston University School of Medicine, Physiology, & Biophysics Boston Massachusetts

W

William Lehman

Boston University School of Medicine, Physiology, & Biophysics Boston Massachusetts

J

Jeffrey R. Moore

Boston University School of Medicine, Physiology, & Biophysics Boston Massachusetts

Funding

National Institutes of Health Award: HL077280

Cite This Article

William M. Schmidt, William Lehman, Jeffrey R. Moore (2015). Direct observation of tropomyosin binding to actin filaments. Cytoskeleton, 72(6), 292-303. https://doi.org/10.1002/cm.21225

Direct observation of tropomyosin binding to actin filaments

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