title: Rotational movement of formins evaluated by using single-molecule fluorescence polarization.
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 FLH, 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.
authors.affiliation: Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life Sciences, Sendai, Miyagi, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizuno
authors.affiliation: Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life Sciences, Sendai, Miyagi, Japan. Electronic address: nwatanabe@m.tohoku.ac.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Watanabe
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004867:Equipment Design; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D058105:Polymerization; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000032:analysis
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate
keywords: Actin filament; Formin homology proteins; Helical rotation; Processive actin polymerization and depolymerization; Single-molecule fluorescence polarization