Fig. 2.

Rng3 promotes motility in vitro by binding actin filaments nonspecifically. (A) SDS gel showing totals (T), supernatants (S), and pellets (P) from a cosedimentation assay in which 700 nM Rng3 and 75 μM actin were pelleted at 400,000 × g for 20 min in 25 mM imidazole, pH 7.4, 46 mM KCl, 23 mM NaCl, 1 mM EGTA, 4 mM MgCl₂, and 2 mM DTT. Rng3 does not pellet in the absence of actin. (B) Panels showing maximum projections of representative fields (128 μm × 128 μm × 50 s) (Movie S2) and average speeds (blue) of motility in 50 mM KCl assay buffer performed in the absence of methylcellulose, to allow diffusion of actin away from the coverslip surface. Rng3 and Myo2 (N-FLAG-C-Biotin) were adhered nonspecifically to nitrocellulose-coated flow cells (15). Motility assay conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, 50 mM KCl, 1 mM EGTA, 4 mM MgCl₂, 10 mM DTT, and 1 mM ATP. Error, ± SD.