Johnston Christopher A, Hirono Keiko, Prehoda Kenneth E, Doe Chris Q
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
Cell. 2009 Sep 18;138(6):1150-63. doi: 10.1016/j.cell.2009.07.041.
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (Pins(LINKER)) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins(TPR) domain has no function alone, but placing the Pins(TPR) in cis to the Pins(LINKER) gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.
不对称细胞分裂受到广泛研究，因为它既能产生细胞多样性，又能维持干细胞群体。不对称细胞分裂需要有丝分裂纺锤体与内在或外在极性线索对齐，但这一过程的机制细节仍不清楚。在此，我们开发了一种方法，用于在正常情况下无极性的细胞系中构建皮质极性，并利用该方法来表征与Inscuteable蛋白的伙伴（Pins；哺乳动物中的LGN/AGS3）相关的纺锤体定向。我们鉴定出一个以前未被认识的进化上保守的Pins结构域（Pins(LINKER)），该结构域需要极光激酶A（Aurora-A）磷酸化才能招募盘状大蛋白（Dlg；哺乳动物中的PSD-95/hDlg）并促进部分纺锤体定向。已被充分表征的Pins(TPR)结构域单独没有功能，但将Pins(TPR)与Pins(LINKER)顺式排列可实现依赖动力蛋白的精确纺锤体定向。这种“诱导皮质极性”检测方法适用于快速鉴定调节纺锤体定向或细胞极性的蛋白质、结构域和氨基酸。
