Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts

Background: The Drosophila central nervous system develops from stem cell like precursors called neuroblasts, which divide unequally to bud off a series of smaller daughter cells called ganglion mother cells. Neuroblasts show cell-cycle-specific asymmetric localization of both RNA and proteins: at late interphase, prospero RNA and Inscuteable, Prospero and Staufen proteins are all apically localized; at mitosis, Inscuteable remains apical whereas prospero RNA, Prospero protein and Staufen protein form basal cortical crescents. Here we use in vitro culture of neuroblasts to investigate the role of intrinsic and extrinsic cues and the cytoskeleton in asymmetric localization of Inscuteable, Prospero and Staufen proteins. Results: Neuroblast cytokinesis is normal in vitro, producing a larger neuroblast and a smaller ganglion mother cell. Apical localization of Inscuteable, Prospero and Staufen in interphase neuroblasts is reduced or eliminated in vitro, but all three proteins are localized normally during mitosis (apical Inscuteable, basal Prospero and Staufen). Microfilament inhibitors result in delocalization of all three proteins. Inscuteable becomes uniform at the cortex, whereas Prospero and Staufen become cytoplasmic; inhibitor washout leads to recovery of microfilaments and asymmetric localization of all three proteins. Microtubule disruption has no effect on protein localization, but disruption of both microtubules and microfilaments results in cytoplasmic localization of Inscuteable. Conclusions: Both extrinsic and intrinsic cues regulate protein localization in neuroblasts. Microfilaments, but not microtubules, are essential for asymmetric protein anchoring (and possibly localization) in mitotic neuroblasts. Our results highlight the similarity between Drosophila, Caenorhabditis elegans, vertebrates, plants and yeast: in all organisms, asymmetric protein or RNA localization and/or anchoring requires microfilaments.