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.