Gαi generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize G alpha i, Pins, and Mud proteins; Pins/G alpha i direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how G alpha i-Pins-Mud binding is regulated to link cortical polarity with spindle orientation. Here, we show that Pins forms a closed state via intramolecular GoLoco-tetratricopepticle repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that Gai binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without opening Pins or recruiting Mud. However, Gai and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopepticle repeat domains, respectively, thereby restricting Pins-Mud interaction to the apical cortex and fixing spindle orientation. We conclude that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation.