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.