Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, 1030 Vienna, Austria.
Mol Biol Cell. 2013 May;24(9):1321-33. doi: 10.1091/mbc.E12-12-0846. Epub 2013 Feb 27.
Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLK(as)) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.
类 Polo 激酶是细胞分裂的重要调节因子，在有丝分裂和细胞骨架遗传中发挥着多样化的作用。在寄生虫布氏锥虫中，单一的 PLK 同源物 TbPLK 对于一系列必需细胞器的组装是必要的，这些细胞器将鞭毛定位并粘附在细胞表面。以前的工作依赖于 RNA 干扰或特异性不明的抑制剂来抑制 TbPLK，这两者都有显著的实验局限性。在这里，我们使用类似物敏感的方法来选择性地和急性地抑制 TbPLK。仅表达类似物敏感的 TbPLK（TbPLK(as)）的 T. brucei 细胞正常生长，但在用抑制剂处理后，鞭毛附着和胞质分裂会出现缺陷。当被抑制时，TbPLK 不能有效地迁移，并在整个细胞周期中被困在细胞的后部。使用同步化的细胞，我们表明活性 TbPLK 是鞭毛附着区组装和延伸的直接要求，鞭毛附着区将鞭毛附着在细胞表面，以及复制的基体旋转的直接要求，基体旋转将新的鞭毛定位，使其能够延伸而不会与旧的鞭毛碰撞。这种方法应该适用于在 T. brucei 基因组中发现的许多缺乏指定功能的激酶。
