Force plays a central role in separating daughter cells during cytokinesis, the last stage of cell division. However, the mechanism of force sensing during cytokinesis remains unknown. Here we discovered that Pkd2p, a putative force-sensing transient receptor potential channel, localizes to the cleavage furrow during cytokinesis of the fission yeast, Schizosaccharomyces pombe. Pkd2p, whose human homologues are associated with autosomal polycystic kidney disease, is an essential protein whose localization depends on the contractile ring and the secretory pathway. We identified and characterized a novel pkd2 mutant pkd2-81KD. The pkd2 mutant cells show signs of osmotic stress, including temporary shrinking, paused turnover of the cytoskeletal structures, and hyperactivated mitogen-activated protein kinase signaling. During cytokinesis, although the contractile ring constricts more rapidly in the pkd2 mutant than the wild-type cells (50% higher), the cell separation in the mutant is slower and often incomplete. These cytokinesis defects are also consistent with misregulated turgor pressure. Finally, the pkd2 mutant exhibits strong genetic interactions with two mutants of the septation initiation network pathway, a signaling cascade essential for cytokinesis. We propose that Pkd2p modulates osmotic homeostasis and is potentially a novel regulator of cytokinesis.

The role of calcium signaling during cytokinesis has long remained ambiguous. Past studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before the cleavage furrow ingression, suggesting that these calcium transients could trigger the contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. Here we explored cytokinetic calcium transients in the fission yeast Schizosaccharomyces pombe by adopting GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level of this model organism. We validated GCaMP as a highly sensitive calcium reporter in fission yeast, allowing us to capture calcium transients triggered by osmotic shocks. We identified a correlation between the intracellular calcium level and cell division, consistent with the existence of calcium transients during cytokinesis. Using time-lapse microscopy and quantitative image analysis, we discovered calcium spikes both at the start of the cleavage furrow ingression and the end of cell separation. Inhibition of these calcium spikes slowed the furrow ingression and led to frequent lysis of daughter cells. We conclude that like the larger animal embryos fission yeast triggers calcium transients that may play an important role in cytokinesis (197). [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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.

Force plays a central role in separating daughter cells during cytokinesis, the last stage of cell division. However, the mechanism of force-sensing during cytokinesis remains unknown. Here we discovered that Pkd2p, a putative force-sensing TRP channel, localizes to the cleavage furrow during cytokinesis of the fission yeast, Schizosaccharomyces pombe. Pkd2p, whose human homologues are associated with Autosomal Polycystic Kidney Disease, is an essential protein whose localization depends on the contractile ring and the secretory pathway. We identified and characterized a novel pkd2 mutant pkd2-81KD. The pkd2 mutant cells show signs of osmotic stress, including temporary shrinking, paused turnover of the cytoskeletal structures and hyper-activated MAPK signaling. During cytokinesis, although the contractile ring constricts more rapidly in the pkd2 mutant than the wild-type cells (50% higher), the cell separation in the mutant is slower and often incomplete. These cytokinesis defects are also consistent with mis-regulated turgor pressure. Lastly, the pkd2 mutant exhibits strong genetic interactions with two mutants of the SIN pathway, a signaling cascade essential for cytokinesis. We propose that Pkd2p modulates osmotic homeostasis and is potentially a novel regulator of cytokinesis.Highlight summary for TOCFission yeast TRP channel Pkd2p is the homologue of human polycystins. The pkd2 mutant exhibits defects in the contractile ring closure and cell separation during cytokinesis. This essential protein localizes to the cleavage furrow where it likely regulates osmotic homeostasis during cytokinesis.

The role of calcium during cell division has long remained ambiguous. The intracellular calcium concentration of many animal embryos increases transiently during cytokinesis, leading to the long-standing proposal that calcium transients may trigger the contraction of actomyosin ring.However, it remains unknown whether these calcium transients can be found in cells beyond those large embryos and whether they have any role in cytokinesis. Here we addressed these questions in the unicellular model organism fission yeast by adopting GCaMP, a genetically encoded indicator, to determine its intracellular calcium level. With confocal microscopy, we captured both the calcium homeostasis and the calcium transients in live cells using this calcium reporter. We searched for the cytokinetic calcium transients using two independent approaches.Both analyses of the intracellular calcium in a population and time-lapse microscopy of dividing cell revealed two cytokinetic calcium spikes. The first initiated at the beginning of the cleavage furrow ingression and the second one at the end of the cell separation. The temporal regulation of these spikes bears a strong similarity to the two calcium waves discovered in the cytokinesis of fish embryos. These slow calcium spikes propagated intracellularly, not restricted to the cell division plane. Although depletion of these spikes did not prevent the contractile ring from contracting as predicted, it reduced the rate of contraction and led to lysis of many daughter cells.We conclude that the fission yeast cytokinetic calcium spikes promote the contractile ring closure and the integrity of separating cells, but they are not required for triggering the ring contraction. Our discovery suggests that transient increase of intracellular calcium may be a conserved regulatory mechanism of cytokinesis among eukaryotes.