Branching MT nucleation amplifies RhoA activation during cytokinesis. (A) The active RhoA reporter Rhotekin is enriched near the MT plus ends of branched MT arrays (top); enlarged view of the branched MT arrays (red dashed box) that activate cortical RhoA (bottom). (B) Fold change in Rhotekin fluorescence intensity near the MT plus ends with respect to a nearby cortical region devoid of astral MTs. Data were pooled from three independent experiments (n = 3). (C) Still frames from a multicolor TIRF time-lapse showing a branching event that locally increases cortical RhoA activation, visualized with Rhotekin. (D) Rhotekin fluorescence increases ∼30% (red) proximal to the mother and daughter MTs during the branching nucleation event shown in C. A nearby cortical region devoid of astral MTs (blue) exhibited no change in Rhotekin fluorescence during the same time period. Red boxes correspond to the time points in C. Error bars indicate SD. Time: min:s. Color wedge, pixel values 150–1,200. Scale bars, 5 µm (A), 1 µm (C).