Formation of an actin axle during the maturation of polarity. During maturation of the polarized form, the circular actin bands around the cell body transitioned to an actin axle along the cell rear. Phase-contrast and AF546-phalloidin FSM time-lapse video of a cell initiating motility and maturing. 30x real time (frames were collected at 3-s intervals and are displayed at 10 frames/s). Phalloidin was conjugated to AlexaFluor546 and cells were imaged in culture media (Leibovitz's L-15 medium without phenol red supplemented with 14.2 mM Hepes, pH 7.4, 10% FBS, and 1% antibiotic-antimycotic). Time-lapse phase contrast and epifluorescent images were acquired using an inverted microscope (Diaphot-300; Nikon) with a 60× NA 1.4 oil plan-Apo objective (Nikon). Images were acquired every 3 s with a 60× oil objective. All time-lapse images were collected with a cooled back-thinned CCD camera (MicroMax 512BFT; Princeton Instruments) with a 2× optovar attached using MetaMorph software version 6 (Molecular Devices). Corresponds to Fig. S3 and video 8 from JCB 178:1207-1221, 2007.
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