Abstract
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.
Highlights
Drosophila neuroblasts (NBs) divide in an asymmetric fashion to generate another NB and a differentiating cell, the ganglion mother cell (GMC) (Lee et al, 2006)
By simultaneously inhibiting Myosin II and Myosin VI, we showed that Myosin II acts upstream of Myosin VI in the same pathway to localize Miranda
Several reports stated that Miranda is apical at interphase and/or at prophase (Fuerstenberg et al, 1998; Matsuzaki et al, 1998; Shen et al, 1998), one report showed that Miranda localizes to the cytoplasm at interphase (Ikeshima-Kataoka et al, 1997)
Summary
Drosophila neuroblasts (NBs) divide in an asymmetric fashion to generate another NB (self-renewal) and a differentiating cell, the ganglion mother cell (GMC) (Lee et al, 2006). The larger, apical daughter cell inherits the self-renewing factors and retains NB fate, whereas the smaller, basal cell inherits differentiation factors and acquires GMC fate (Barros et al, 2003; Yu et al, 2006). Adaptor proteins, such as Miranda and Partner of Numb (PON), play a pivotal role in asymmetric cell division because they ensure the asymmetric segregation of cell fate determinants to the GMC (Betschinger and Knoblich, 2004). PON binds to and localizes Numb, a membrane-associated protein and a negative regulator of Notch signaling, to the basal cortex (Lu et al, 1998)
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