Prolonged induction of VPS8D hairpin expression leads to loss of CVC structures
Following longer periods of VPS8D -hairpin induction, the CVC and associated markers underwent more dramatic changes. Strikingly, many cells viewed with DIC optics lacked any visible contractile bladders, strongly suggesting that the bladders are lost during sustainedVPS8D knockdown. Not surprisingly, all CVC proteins in such cells showed a marked change in their distribution compared to in wildtype. Dop1p no longer localizes to a recognizable bladder but is instead dispersed throughout the cytoplasm including in many small puncta (Fig. 3A). Vma4p is also dispersed throughout the cell, visible as diffuse fluorescence but also present in large, potentially multi-vesicular structures (Fig. 3B). A second protein that localizes to the spongiome in wildtype cells, Scr7p, is also delocalized after extendedVPS8D -hairpin induction. The delocalization of these two spongiome markers is likely to reflect the loss of the spongiome, in parallel to the loss of the bladder.
Taken together, these results strongly suggest that the maintenance of multiple compartments of the CVC depend on Vps8Dp. The spongiome may be more acutely dependent on Vps8Dp-assisted membrane trafficking than the bladder.