Microtubules position the leading edge across from the actomyosin-enriched rear. (A) TIRF microscopy time-lapse series of a cell expressing RLC-D,D–GFP (localized at the rear, marked by arrowheads) in the presence of the microtubule inhibitor nocodazole (5 µM). Note that the direction of movement of the leading protrusion (arrow) is bent (almost perpendicular to the major polarity axis, indicated by a dashed line), and the leading protrusion has separated from the rear; the asterisks point to the site of ripping. The complete movie is shown in Video 3. Bar, 10 µm. (B) Cells expressing RLC-D,D–GFP and migrating on fibronectin in the presence or absence of 5 µM nocodazole (NCD) or 0.1 µM vinblastine (VIN). Axis ratio is calculated as the ratio between long (migratory) and short (perpendicular, passing through the center of the nucleus) axes; thus, an AR of 1 (indicated with a horizontal line) denotes a round cell. Error bars indicate ±SD. (C) Diagrams showing the relative direction of the leading protrusion with respect to the axis established by the extended rear in control and 5 µM nocodazole– or 0.1 µM vinblastine–treated cells. n > 100 cells were quantified from two independent experiments.