Department of Biological Sciences, University of Toledo, Toledo, OH 43606.
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109.
Mol Biol Cell. 2022 Dec 1;33(14):ar134. doi: 10.1091/mbc.E22-07-0248. Epub 2022 Oct 6.
Pkd2 is the fission yeast homologue of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of mutant cells. Pkd2 was translated and inserted into the lipid bilayers of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypoosmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation reduced the amplitude of hypoosmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of reduced the calcium spikes, accompanying cell separation and the ensuing membrane stretching, by 60%. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.
Pkd2 是裂殖酵母多聚蛋白的同源物。这个假定的离子通道定位于质膜。它在细胞间期生长和胞质分裂期间，即细胞分裂的最后一步，细胞体积的扩张中是必需的。然而，Pkd2 的通道活性尚未得到测试。在这里，我们通过体外重构和突变细胞的钙成像来检查 Pkd2 的钙通透性和机械敏感性。使用无细胞表达系统翻译并将 Pkd2 插入脂质双层中。当通过低渗冲击拉伸膜时，重构的 Pkd2 渗透钙。在体内，通过温度敏感突变 失活 Pkd2 可使细胞内平均钙水平降低 34%。与野生型相比，低功能突变 使低渗冲击触发的钙峰幅度降低了 59%。在胞质分裂期间，突变 使钙峰降低了 60%，伴随细胞分离和随后的膜拉伸。我们得出结论，裂殖酵母多囊蛋白 Pkd2 允许钙流入，当被膜拉伸激活时，代表一种可能的机械敏感性通道，有助于胞质分裂钙峰。
