Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI

The adaptor protein Miranda plays a pivotal role in the asymmetric cell division of neuroblasts by asymmetrically segregating key differentiation factors. Miranda localization requires Myosin VI and Myosin II. The apical-then-basal localization pattern of Miranda detected in fixed tissue, and the localization defects in embryos lacking Myosin VI, suggest that Miranda is transported to the basal pole as a Myosin VI cargo. However, the mode and temporal sequence of Miranda localization have not been characterized in live embryos. Furthermore, it is unknown whether Miranda and PON, a second adaptor protein required for asymmetric protein localization, are both regulated by Myosin II. By combining immunofluorescence studies with time-lapse confocal microscopy, we show that Miranda protein forms an apical crescent at interphase, but is ubiquitously localized at prophase in a Myosin-II-dependent manner. FRAP analysis revealed that Miranda protein reaches the basal cortex by passive diffusion throughout the cell, rather than by long-range Myosin VI-directed transport. Myosin VI acts downstream of Myosin II in the same pathway to deliver diffusing Miranda to the basal cortex. PON localization occurs mainly along the cortex and requires Myosin II but not Myosin VI, suggesting that distinct mechanisms are employed to localize different adaptor proteins during asymmetric cell division.