Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Dev Biol. 2012 May 1;365(1):219-28. doi: 10.1016/j.ydbio.2012.02.027. Epub 2012 Feb 25.
During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.
在不对称的干细胞分裂过程中，细胞皮层的极化将命运决定因素不均匀地靶向到姐妹细胞中，导致干细胞的再生和具有有限发育潜力的祖细胞的产生。在果蝇幼虫大脑中的有丝分裂神经干细胞（神经母细胞）中，极性蛋白致死（2）巨幼虫（Lgl）和非典型蛋白激酶 C（aPKC）之间的拮抗相互作用通过调节命运决定因素的不对称分离，确保一个女儿神经母细胞的自我更新和祖细胞的产生。在 lgl 功能缺失的情况下，升高的皮层 aPKC 激酶活性扰乱了命运决定因素（包括 Numb）的不均匀分配，并在幼虫脑中诱导了过多的神经母细胞。然而，增加的 aPKC 功能是否通过使 Numb 失活来触发过多的神经母细胞的形成仍存在争议。为了研究增加的皮层 aPKC 功能如何诱导过多的神经母细胞的形成，我们分析了 lgl 突变体大脑中神经母细胞谱系克隆中细胞的命运。令人惊讶的是，我们的分析表明，lgl 突变体大脑中的神经母细胞经历不对称分裂以产生祖细胞，然后祖细胞再变回神经母细胞。在 lgl 突变体大脑中，Numb 仍然定位于有丝分裂神经母细胞的皮层中，并且在不对称分裂完成后不能将其完全分离到祖细胞中。这些结果使我们提出，皮层中升高的 aPKC 功能降低了未来祖细胞中 Numb 的功能。我们确定酰基辅酶 A 结合域包含 3 蛋白（ACBD3）结合区域对于 Numb 在有丝分裂神经母细胞中的不对称分离以及增加的 aPKC 功能诱导的多余神经母细胞表型的抑制是必不可少的。Numb 的 ACBD3 结合区域包含两个 aPKC 磷酸化位点，丝氨酸 48 和 52。令人惊讶的是，虽然这两个位点的磷酸化状态直接影响有丝分裂神经母细胞中 Numb 的不对称分离，但 Numb 的磷酸化和非磷酸化形式都抑制了由增加的皮层 aPKC 功能触发的多余神经母细胞的形成。因此，我们提出，皮层 aPKC 激酶活性的精确调节部分通过确保将 Numb 不对称地分配到未来的祖细胞中，从而区分姐妹细胞的身份，在那里 Numb 独立于 aPKC 的调节而保持有限的潜力。
