VPS8D knockdown affects features of both the bladder and spongiome
Conventional CORVET in S. cerevisiae acts as a determinant in the maturation of endosomal compartments (Peplowska et al., 2007). To ask whether knockdown of T. thermophila VPS8D led to changes in the CVC, we generated cells expressing the VPS8D hairpin in combination with endogenously tagged copies of either Dop1p, which most strongly labels the bladder, or tagged Vma4p which decorates the spongiome (Cheng et al., 2023).
Following 5 hours of hairpin induction, Dop1p-mNeon still shows clear localization to the CV bladder that is qualitatively similar to that in non-induced or wildtype cells, and the bladder profiles are also similar (Fig. 2A, compare left and right panels). However, by 8 hours of hairpin induction the intensity of Dop1p bladder steady state labeling was significantly decreased (Fig. 2B and S2). This change in the association between Dop1p and the bladder membrane upon VPS8D knockdown could also be seen at the level of dynamics. We previously used fluorescence recovery after photobleaching (FRAP) to discover that Dop1p at the bladder exchanges with a cytosolic pool (Cheng et al., 2023), as shown in Figure 2C and 2D. That exchange was inhibited in VPS8Dknockdown cells, so that the rate of fluorescence recovery at the bleached bladder was decreased (Fig. 2E, 2F and 2G). Lastly, the period between contractions was lengthened in these VPS8D knockdown cells (Fig. 2H). The mechanisms controlling bladder contraction are not known, but one possibility is that bladder refilling is less efficient in the mutant. Intriguingly, VPS8D hairpin induction had a clear effect on the spongiome morphology. In particular, the volume occupied by Vma4p-decorated tubules showed a marked decrease within 5 hours ofVPS8D -hairpin induction (Fig. 2I, 2J and S3), suggesting that spongiome architecture depends more acutely than bladder structure on the activity of Vps8Dp. Interestingly, these rapid changes in spongiome structure upon VPS8D knockdown are similar to those seen after cells are exposed to brief hypoosmotic challenge (Fig. S4).