Fig. 4.

Localization and polymerization dynamics of cp-actin filaments. (A) Three dimensional reconstruction of a chloroplast (shown with red chlorophyll autofluorescence) using photographs taken every 0.1 μm in the z axis by confocal microscopy and cp-actin filaments (shown with green fluorescence of GFP-talin). Distribution of cp-actin filaments at the plasma membrane side of chloroplasts (which is obvious from chloroplast shape) both in nonbiased and biased cp-actin filaments (Movie S10 and Movie S11, respectively). (B) Disappearance of cp-actin filaments under a high-intensity laser beam (1 mW) for GFP excitation (Movies S13). (C) Disappearance followed by reappearance of cp-actin filaments under a low-intensity laser beam (0.4 mW) (Movie S14). (D) Cp-actin filament images taken every 3 s (b1 and b2) or 10 s (c1) in rectangles B and C (see Movie S14) (E) Kymographs representing cp-actin disappearance (made of 120 photographs taken every 0.5 s on the white lines in b1 and b2 of B) and reappearance (made of 180 photographs taken every 1 s on the white lines in c1 and c2 of C) at the chloroplast edge. Arrows show chloroplast edges. The outside of the chloroplast is on the left. (F) Repeated appearance of cp-actin filaments at the same points of the chloroplast edge (shown with arrowheads). A protoplast was repeatedly observed by fluorescence microscopy for 30 s followed by 1–2-min incubation in the dark. Photographs were taken at the beginning (1, 3, 5, and 7) and ending (2, 4, 6, and 8) of the observation period. A 30-s observation caused cp-actin filament disappearance and a 1–2 min dark treatment allowed the recovery.