Fig. 1.

KIF13B transport of ADAP1 is inhibited by PIP₃. (A) Domain architecture of ADAP1 and KIF13B and known interactions (Top). Architecture of protein constructs used in this study (Bottom). CC, coiled coil; CG, cytoskeleton-associated protein glycine-rich domain; FHA, forkhead-associated domain; MBS, membrane-associated guanylate kinase-binding stalk. (B) Assay scheme for single-vesicle motility assay. Fluorescently labeled vesicles are observed by TIRF microscopy as they are transported along microtubules by molecular motors. (C, Left) Time series of rhodamine-labeled NTA vesicles (green; 3 mol % Ni-NTA) being transported along HiLyte 647-labeled microtubules (red) in the presence of 500 nM KIF13B-6×His (Top) and corresponding time average (100 frames) of the rhodamine channel (Bottom). (C, Right) Time series of rhodamine-labeled PIP₃ vesicles (green; 5 mol % PIP₃) in the presence of ADAP1 (here 500 nM) and KIF13B (here 500 nM) and corresponding time average (100 frames) of the rhodamine channel (Bottom). (D) Quantification of run frequencies corresponding to C counting run events per minute and per micrometer of microtubule with one representative kymograph per condition. ***P < 0.001. (E) Assay scheme for single-molecule motility assay. Fluorescently labeled proteins are observed by TIRF microscopy along surface-immobilized microtubules. (F, Top) Time series of CF₄₈₈-labeled KIF13B (green; 10 nM) walking along HiLyte 647-labeled microtubules (red) in the presence of 23 μM water-soluble PIP₃-C4. (F, Bottom) Corresponding time average of the 488-nm channel (total duration 100 frames). (G, Top) Time series of 20 nM mWasabi-ADAP1 (green) with 100 nM unlabeled KIF13B in the absence (Left) or presence (Right) of 23 μM water-soluble PIP₃-C4. (G, Bottom) Corresponding time average of the 488-nm channel (total duration 100 frames). (H) Quantification of run frequencies corresponding to G with one representative kymograph of the 488-nm channel per condition. ***P < 0.001. Displayed box plots encompass the 25–75th percentiles, the midline indicates the median, the whiskers extend to show the rest of the distribution and to indicate outliers.