Membrane stretching activates calcium permeability of a putative channel Pkd2 during fission yeast cytokinesis.

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 pkd2 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 pkd2-B42 reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypoosmotic shock–triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 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是多囊蛋白在裂殖酵母中的同源物。这种假定的离子通道定位于质膜。它在间期生长和胞质分裂（细胞分裂的最后一步）过程中细胞体积的扩张是必需的。然而，Pkd2的通道活性尚未经过测试。在此，我们通过体外重组以及对pkd2突变细胞的钙成像来检测Pkd2的钙通透性和机械敏感性。利用无细胞表达系统，Pkd2被翻译并插入到巨大单层囊泡的脂质双层中。当通过低渗冲击使膜拉伸时，重组的Pkd2可使钙通透。在体内，通过温度敏感突变pkd2 - B42使Pkd2失活，使平均细胞内钙水平降低了34%。与野生型相比，亚效等位基因突变pkd2 - 81KD使低渗冲击触发的钙峰值幅度降低了59%。在胞质分裂过程中，pkd2的突变使伴随细胞分离和随后的膜拉伸的钙峰值降低了60%。我们得出结论，裂殖酵母多囊蛋白Pkd2在被膜拉伸激活时允许钙内流，代表了一种可能的机械敏感通道，它对胞质分裂钙峰值有贡献。