Moesin directly anchors microtubules to the cortex and regulate their dynamics. (A) An illustration depicts a proximity ligation in situ assay (PLA; Duolink), which allows the detection of protein–protein interactions as bright fluorescent spots in situ. (B) MoesinT₅₅₉D-GFP (MoeT₅₅₉D) interacts with microtubules at the cortex of S2 cells, as evident by the presence of fluorescent Duolink spots at the periphery of the cell (red, top). Interphase cells plated on glass are shown. MT, microtubule. (C) Quantification of Tubulin/Moesin-GFP Duolink spots in cells expressing the indicated constructs. Boxes show top and bottom quartiles, horizontal lines show median values, and vertical lines show minimal and maximal values (control, n = 12; MoesinT₅₅₉D, n = 12; MoesinK_212,213M,T₅₅₉D, n = 10). (D) Time-lapse projection of Tubulin-GFP in S2 cells transfected with MoesinK_212,213M,T₅₅₉D-mCherry construct. Heat map representation is shown as in Fig. 1. (E) Ends of GFP-labeled microtubules were tracked using an automated tracking algorithm. The microtubule ends in MoesinT₅₅₉D-expressing cells (two cells; 203 microtubules) spent significantly more time in the vicinity of the cortex compared with control cells (three cells; 310 microtubules) or cells expressing MoesinK_212,213M,T₅₅₉D-GFP (two cells; 164 microtubules). Bars: (B) 10 µm; (C and D) 5 µm.