Figure 7.

Rng2Ns and Fim1 stabilize actin filaments against Adf1-induced depolymerization. (A) Localization of GFP-Adf1. Cells expressing GFP-Adf1 were stained for DNA. (B) Time course of the depolymerization of F-actin after dilution in the presence of Adf1. G-actin (3 μM, 20% pyrene labelled) plus 15 nM gelsolin was polymerized with Rng2Ns or Fim1. F-actin was diluted to 0.1 μM with 0.11 μM Adf1, and the decrease in pyrene fluorescence was monitored. (C) Data in B were fitted to single exponentials, and the disassembly rate was plotted as a function of Rng2Ns or Fim1 concentration. (D–G) Fluorescence micrographs of actin filaments during depolymerization. F-actin (3 μM in G-actin) plus 15 nM gelsolin was mixed with 1.8 μM Rng2Ns (F) or Fim1 (G) for 1 h, diluted 30-fold with 0.12 μM Adf1, removed from the reactions at the indicated time points after dilution, and stained with rhodamine–phalloidin. F-actin plus gelsolin alone was also diluted in the absence (D) or presence (E) of Adf1. (H, J) Co-sedimentation of Rng2Ns or Fim1 with F-actin prebound to Adf1. F-actin (2 μM) was mixed with 4 μM Adf1 (+Adf1) or buffer (–Adf1) for 30 min at pH6.0 or 8.0 (track 7 in H) and then mixed with 1 μM Rng2Ns or Fim1, before being centrifuged at (H) high speed or (J) low speed. The proteins in the supernatants (S) and pellets (P) were resolved by SDS–PAGE and CBB-stained. (I, K) The co-sedimentation assays shown in (H) and (J) were performed for various concentrations of Adf1. The molar ratios of Rng2Ns or Fim1 to actin in the pellet produced by high-speed sedimentation (I) or the amount of actin in the pellet produced by low-speed sedimentation (K) was normalized to that in the absence of Adf1 and plotted versus the Adf1/actin concentration ratio. Data are expressed as the means±s.d. (error bars) of three independent experiments. Bars: 5 μm.