VPS8D knockdown results in a proliferation of large
vesicles or vacuoles bearing CV markers
If Vps8Dp as part of a CORVET complex is active in promoting fusion
between membranes, then the disappearance of CVC structures uponVPS8D knockdown may be due to a fusion defect between
intermediates in CVC formation. To explore this idea, we analyzed cells
that showed intermediate CVC phenotypes after induction of theVPS8D hairpin. In these experiments, cells were treated with low
concentrations of cadmium and for short time periods. In such samples,
it was easy to find cells displaying CVC features never seen in wildtype
cells, with examples in Figures 4A (wildtype) vs the knockdown cells
(Fig. 4B and 4C). The cell shown in panel B possesses what appear to be
two adjacent CV bladders. These persist for tens of seconds before one
of them contracts rapidly (t = 28sec), which is then followed by
contraction of the second bladder at 29 sec. This asynchronicity implies
that the bladders are linked with two different plasma membrane pores.
Over the next 20 seconds, just one of the bladders appears to refill. In
panel C, the cell contains at least seven medium sized-to-large
Dop1p-labeled vesicles. Over the course of the 15 seconds in the video,
one of the two largest vesicles contracts, followed shortly thereafter
by the second. Within 5-6 seconds, both show signs of refilling and also
appear tightly associated with smaller labeled vesicles at their
periphery. The Dop1p-labeled relatively immobile vesicles in this cell
are spread over a large volume of the cell posterior, so that most
cannot be closely associated with CV pores. The presence in a large
subset of the knockdown cells of heterogeneous Dop1p-labeled
compartments, which include multiple contractile bladders, may be
consistent with the idea that maintenance of the CVC in wildtype cells
requires Vps8Dp-dependent membrane fusion.