Pkd2 is the fission yeast homolog 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 the pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayer of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypo-osmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared to the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypo-osmotic 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.

No abstract

Pkd2 is the fission yeast homolog of polycystins. This putative ion channel localizes to the plasma membrane and 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 the pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayer of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypo-osmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared to the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypo-osmotic shock-triggered calcium spikes by 59%. This pkd2 mutation also reduced the long-term adaption of fission yeast to hypo-osmotic shock through nuclear translocation of the transcription factor Przl. We concluded that the fission yeast polycystin Pkd2 is calcium-permeable when activated by membrane stretching, likely representing a novel eukaryotic mechanosensitive channel that can sense membrane tension and activate calcium signaling pathways.

Lower temperatures delayed development of systemic symptoms by Cauliflower mosaic virus (CaMV) in two different plant hosts. However, lower temperature exposure increased CaMV nucleic acid levels in leaves of systemically-infected turnips. Furthermore, lower temperature altered the formation of aggregates formed by the CaMV major inclusion body (IB) protein, P6. Finally, lower temperature altered the architecture of the actin cytoskeleton. These data may suggest that lower temperatures alter the actin cytoskeleton, facilitating the formation of larger IBs that hold on to their internal virions more strongly than small ones, impairing virus particle release and causing a delay in systemic infection.