Fig. 6. FRAP of F-actin in cones to examine actin dynamics. (A,B) FRAP experiment 1. Line bleaching across actin cones in a moving cystic bulge. (A) Live image of a moving cystic bulge from a GFP-actin expressing cyst. Arrowhead indicates position of the bleached actin cones. (B) Time-lapse recording of fluorescence recovery after photobleach across a single actin cone. The cystic bulge moved from the bottom to the top of the photograph. Arrowhead indicates position of photobleach. The numbers indicate time after photobleach. Fluorescence recovered completely between 7 minutes 21 seconds and 9 minutes 48 seconds time-points. The bleached spot moved forward as the actin cone moved forward. (C,D) FRAP experiment 2. Bleaching and recovery in a whole single actin cone. (C) Actin cones in moving cystic bulge. Arrowhead indicates actin cone to be bleached. (D) Time series of photobleach and recovery of a whole single actin cone. Recovery takes place relatively uniformly along the cone, and plateaued after 17 minutes 9 seconds, although the fluorescence intensity did not recover completely. (E) FRAP experiment 3. Time series of bleaching and recovery in very early actin cones before onset of movement. Half of the actin cone cluster was bleached. The fluorescence intensity plateaued after 43 minutes 36 seconds. Scale bars: 10 µm for A,C,E; 2.5 µm for B,D.

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