Broadus J, Doe C Q
Department of Cell and Structural Biology, Howard Hughes Medical Institute, University of Illinois, Urbana 61801, USA.
Curr Biol. 1997 Nov 1;7(11):827-35. doi: 10.1016/s0960-9822(06)00370-8.
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.
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.
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.
果蝇中枢神经系统由称为神经母细胞的干细胞样前体细胞发育而来，神经母细胞进行不均等分裂，产生一系列较小的子细胞，即神经节母细胞。神经母细胞在细胞周期的特定阶段会出现RNA和蛋白质的不对称定位：在分裂间期晚期，prospero RNA以及Inscuteable、Prospero和Staufen蛋白均定位于顶端；在有丝分裂期，Inscuteable仍位于顶端，而prospero RNA、Prospero蛋白和Staufen蛋白则形成基部皮质新月体。在此，我们利用神经母细胞的体外培养来研究内在和外在信号以及细胞骨架在Inscuteable、Prospero和Staufen蛋白不对称定位中的作用。
神经母细胞的胞质分裂在体外是正常的，产生一个较大的神经母细胞和一个较小的神经节母细胞。Inscuteable、Prospero和Staufen在分裂间期神经母细胞中的顶端定位在体外会减少或消失，但这三种蛋白在有丝分裂期间的定位正常（Inscuteable位于顶端，Prospero和Staufen位于基部）。微丝抑制剂会导致这三种蛋白的定位发生改变。Inscuteable在皮质上变得均匀分布，而Prospero和Staufen则进入细胞质；去除抑制剂后，微丝恢复，这三种蛋白的不对称定位也得以恢复。微管破坏对蛋白定位没有影响，但微管和微丝同时被破坏会导致Inscuteable定位于细胞质中。
内在和外在信号均调控神经母细胞中的蛋白定位。微丝而非微管对于有丝分裂期神经母细胞中蛋白的不对称锚定（可能还有定位）至关重要。我们的结果凸显了果蝇、秀丽隐杆线虫、脊椎动物、植物和酵母之间的相似性：在所有生物体中，蛋白或RNA的不对称定位和/或锚定均需要微丝。
