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.