RNAi-mediated knockdown of VPS8D results in defects consistent with a role at the osmoregulatory CVC
The role of Vps8Dp at the CVC is not known, but the gene appeared to be essential since we could not recover viable cells when pursuing a gene knockout strategy (Sparvoli et al., 2018). We therefore chose to pursue a gene knockdown approach, by constructing an RNA hairpin corresponding to a part of the VPS8D gene (Fig. 1B) and expressing it inTetrahymena under the control of the cadmium-inducibleMTT1 promoter (Shang et al., 2002, Howard-Till and Yao, 2006). The effectiveness of such hairpins in T. thermophila can be difficult to assess at the level of the targeted transcripts (Howard-Till and Yao, 2006). To circumvent this issue, we directly measured protein levels by generating a cell line in which Vps8Dp was endogenously tagged with the FLAG epitope, and the VPS8D hairpin was inducibly expressed from the RPL29 gene locus (Yao and Yao, 1991) (Fig. 1C). Induction of the hairpin expression led to a significant decrease in the level of Vps8Dp-FLAG, as measured by immunofluorescence (Fig. 1D). Importantly, after hypoosmotic shock, the knockdown cells displayed morphological changes consistent with a defect in maintaining osmotic homeostasis, losing their teardrop shape and becoming roughly spherical (Fig. 1E) Equivalently-treated wild type cells could quickly recover their normal shape (Fig. 1E). Similarly, while the wildtype cells retained full viability after the osmotic challenge, the VPS8D knockdown cells died shortly thereafter (Fig. 1F). The differences in the cell responses after hypoosmotic shock between wild type and VPS8D -KD cells are diagrammed in Fig. 1G. Under these stress conditions, the activity of the CVC appears critical not to prevent osmotic swelling but to allow recovery after such events.