Amot interaction with Yap1 is required for proper growth of dendritic arbors.

(A) Schematic illustration of Amot protein with mutated Yap1-binding sites where PPXY motifs were modified into PPXA. (B) Rat hippocampal neurons that were transfected on DIV7 with the indicated constructs. (C) Quantification of TDL of Amot-knockdown rat hippocampal neurons that were cotransfected with plasmids as in B. Control cells were transfected with an empty pLKO vector. Control: n = 107; Amot shRNA: n = 80; Amot shRNA + Amot: n = 100; Amot shRNA + Amot ΔYap1: n = 59. To control; p p p = 0.3853. Images were obtained from at least from three independent cultures. (D) Yap1 phosphorylation at serine-127 was visibly reduced in Amot-depleted neurons, whereas the total cellular level of Yap1 was unchanged. (E) Yap1 coprecipitated with Amot from mouse brain homogenates. Uncoated magnetic beads were used as the control. (F) Western blot analysis of Yap1 expression in different regions of the mouse brain. “OB” = olfactory bulbs; “Mb” = midbrain; “Hipp” = hippocampus; “Th” = thalamus; “Cx” = cortex; “Cb” = cerebellum. (G, H) Yap1 localization in the brain, showing cryostat sections of the P30 mouse hippocampus (G) and cerebellum (H) that were stained for Yap1 (green) and Syn (red). Overlay images additionally showed nuclei, visualized with DAPI (blue in G), and Purkinje cell dendritic processes, visualized with anti-calbindin antibody (blue in H). (I) Yap1 expression increased at later stages of neuronal development. Neuronal extracts that were collected on the indicated DIVs were analyzed by western blot. (J) Yap1 localized to dendritic processes in young cultured neurons. Rat hippocampal neurons (DIV8) were immunolabeled for Yap1 (green) and Map2 (red). (K) Yap1 localized to synapses in mature cultured neuronal cells. Rat hippocampal neurons (DIV21) were immunolabeled for Yap1 (green) and Syn (red). The lower panels in G, H, and K show higher magnifications of the boxed areas in the upper panels. Scale bars = 50 μm in B; 10 μm in G, H, J, and K (upper panel); and 5 μm in K (lower panel). Numerical values that underlie the graphs are shown in S1 Data. Statistical significance was analyzed using one-way analysis of variance followed by Tukey’s post hoc test. ****p < 0.0001. Bars represent the mean ± SEM. AB, actin-binding motif; CC, coiled-coil domain; DIV, day in vitro; ID, intrinsic disordered; IP, immunoprecipitation; Map2, microtubule-associated protein 2; ns, not significant; P, postnatal day; PR, proline-rich region; SEM, standard error of the mean; shRNA, short-hairpin RNA; Syn, synaptophysin; TDL, total dendrite length; Yap1, Yes-associated protein 1.