Moesin binds microtubules in vitro and regulates their cortical dynamics in cultured cells. (A) Time-lapse projection of microtubules in Tubulin-GFP cells transfected with mCherry (left) or MoesinT₅₅₉D-mCherry (right). (top) Time frames of the Tubulin-GFP channel is overlaid using heat map (blue to red, 60 frames every 5 s). The asterisk indicates a nontransfected cell, and arrows show representative microtubules used for kymography (bottom). (bottom) Kymographs of microtubules. The cell edge is outlined in red. (B) A plot showing the percentage of time spent by individual microtubules at the cortex (<500 nm from the cell edge). Boxes show top and bottom quartiles, horizontal lines show median values, and vertical lines show minimal and maximal values. (C) Constructs were titrated against increasing concentrations of microtubules (MT) in a cosedimentation assay. Coomassie blue–stained gels of supernatant (S) and pellet (P) fractions are shown. (D) Fractions of microtubule-bound Moesin from four independent cosedimentation experiments were plotted against microtubule concentrations and fitted to a hyperbola to determine the K_d. Error bars represent SDs. (E) An illustration depicting the microscopy-based microtubule-binding assay. Moe, Moesin. (F) Microscopy images of X-rhodamine–microtubules captured by surface-anchored GST (left) or GST-Moesin_1–559 (right). Bars: (A [top] and F) 5 µm; (A, bottom) 1 µm.