Figure 2. iPAK4 fibers can be sequentially labeled with different dyes.

a, Scheme for multi-color labeling of intracellular iPAK4 fibers. b, Images of iPAK4 fibers labeled with four dye transitions on each end. Triangles indicate times of dye addition. Blue: JF₅₀₃, Red: JF₆₆₉, Yellow: JFX₆₀₈. Scale bars 5 μm. c, Comparison of the growth rate on the two ends after one dye switch. The long end and short end correspond to the faster and slower growing end, respectively (N = 153 fibers). The growth rate on the slower-growing end was 0.77 ± 0.05-fold lower than on the faster-growing end (95% C.I.). d, Comparison of two ends of a single fiber. Top: images of the two ends. In this case, the fiber split at one of its ends, an occurrence in approximately 10% of fibers. Bottom: quantification of the fluorescence traces in the two ends, with position normalized to the locations of the first JF₆₆₉ addition and the end of the fiber. Scale bar 5 μm. e, Comparison of dye transition points on the faster and slower-growing fiber ends. The scatter plot shows the positions of the JFX₆₀₈, JF₅₀₃ and JF₆₆₉ transitions normalized relative to the first JF₆₆₉ addition (at position 0) and the end of the fiber (at position 1). The plots show the mean fluorescence profiles of N = 17 fibers. Here the number of fibers was set by the requirement for in-focus, unobstructed views of both ends of the fibers. f, Mean fluorescence profiles of fibers exposed to the same sequence of dyes and then grown for different amounts of time (N = 22 fixed at 8 h, 12 fixed at 30 h). The overlap of these profiles indicates negligible monomer exchange over 22 h.