Rotational movement of formins evaluated by using single-molecule fluorescence polarization

Hiroaki Mizuno, Naoki Watanabe

    Research output: Chapter in Book/Report/Conference proceedingChapter

    5 Citations (Scopus)

    Abstract

    Formin homology proteins (formins) are responsible for the formation of actin structures such as actin stress fibers, actin cables, and cytokinetic contractile rings. Formins are the major actin filament (F-actin) nucleators in the cell. Because formins remain bound to the barbed end after nucleating an actin filament, it was expected that formins might rotate along the double-helical structure of F-actin during processive actin elongation (helical rotation). Here, we describe a method to detect the rotational movement of F-actin elongating from immobilized formins using single-molecule fluorescence polarization (FLP). Tetramethylrhodamine (TMR) attached to Cys-374 of actin emits polarized fluorescence at ≈ 45 with respect to the filament axis. When the TMR-labeled F-actin laying at 45 in the visual field rotates, the vertical- and horizontal-polarized fluorescence (FLV and FL H, respectively) of TMR alternately become bright. This technique allowed us to demonstrate the helical rotation of mDia1, a mammalian formin. Adenosine triphosphate (ATP) hydrolysis in actin subunits is not required for helical rotation; however, ATP appears to contribute to accelerating actin elongation by mDia1. When helical rotation is limited by trapping both mDia1 and the pointed-end side, the processive filament elongation is blocked. Thus, mDia1 faithfully rotates along the long-pitch helix of F-actin. In this chapter, we introduce the theoretical concept of single-molecule FLP, the optical setup, the preparation of adenosine diphosphate-bound actin, and the procedure to observe the rotational movement of F-actin elongating from immobilized formins.

    Original languageEnglish
    Title of host publicationReconstituting the Cytoskeleton
    PublisherAcademic Press Inc.
    Pages73-94
    Number of pages22
    ISBN (Print)9780123979247
    DOIs
    Publication statusPublished - 2014 Jan 1

    Publication series

    NameMethods in Enzymology
    Volume540
    ISSN (Print)0076-6879
    ISSN (Electronic)1557-7988

    Keywords

    • Actin filament
    • Formin homology proteins
    • Helical rotation
    • Processive actin polymerization and depolymerization
    • Single-molecule fluorescence polarization

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

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