EB3-GFP shows steady MT dynamics ex vivo and in vivo, whereas GFP-tubulin highlights a durable MT frame. A single image, focused near the surface of a plated fiber expressing EB3-GFP (A1), shows typical puncta. The dynamics can be appreciated in the corresponding time-lapse series (Video 1) and in its projection (A2). Color coding of the projection helps to visualize movement: the first image of the series is colored blue and the last one magenta, as in the bar. EB3-GFP puncta mostly move longitudinally and transversely, as if along parallel and antiparallel tracks (A2, arrows). Some MT intersections (arrowheads) seem to behave as MT nucleation sites. An image from another fiber (B1) and the kymograph of the line between the arrowheads (B2; see Materials and methods) indicate that puncta move at the same speed in either direction on the longitudinal axis (oblique lines; red arrows). EB3-GFP dynamics in vivo (C1 and C2; Videos 2 and 5 and Fig. S2) validate plated fibers in all respects (arrowheads point to nucleation sites). In contrast, GFP-tubulin in plated fibers (D–G) appears static: the color-coded projections (D2, F1, and F2), with the same number of images and frame rate as A2, are practically white. An aster (arrowhead) indicates an MT nucleation site. The kymograph (E2) of the line between the arrowheads (E1) shows stationary MTs, with occasional local movement (arrows). Muscle MTs show dynamic instability (G); the asterisk shows the plus end of a MT growing and shrinking over 80 s. See Tables 1 and S1 for data quantitation and technical parameters. Bars: (A–E) 10 µm; (insets) 2 µm; (kymograph vertical time axes) 60 s.