VPS8D is required for features of the bladder and spongiome.
A: Dop1p localization becomes more dispersed upon VPS8Dknockdown. Growing cells (WT or VPS8D- KD) expressing Dop1p-mNeon at the DOP1 locus were imaged to assess Dop1p localization. WT (left panel); induced VPS8D knockdown (right panel). Images were taken with a Zeiss Axio Observer 7 system.
B: Dop1p localization to the bladder decreases upon VPS8Dknockdown. VPS8D -KD cells expressing Dop1p-mNeon were incubated without (-Cd) or with (+Cd) cadmium for 3,5 or 8 hours and then fixed for imaging. 20 images for each sample were analyzed to quantify Dop1p fluorescence at the bladder and for the whole cell (Fig. S2). The data were plotted using GraphPad Software Prism and analyzed using a two-tailed t -test. The ratios of the mean±s.d. intensities of Dop1p fluorescence at the bladders relative to the whole cells from each sample are shown. The difference between +Cd and -Cd after 8-hour treatment is significant, P -value <0.0001.
C-G: Dop1p exchange kinetics are slowed upon VPS8D knockdown.
C. Wild type cell expressing Dop1p-mNeon analyzed by FRAP. Growing cells were immobilized and live imaged with a Marianas spinning disc confocal microscope with a FRAP tool. Three images were extracted from a video (Movie 1) showing a photobleaching event and recovery. Left panel: cell at 11s, immediately before photobleaching. Middle panel: cell at t=12s immediately after photobleaching a region containing part of the CV bladder. The bleached area appears as a dark square area outlined with a red dotted box. Right panel: cell at t=2 min 1s.
D: The recovery after photobleaching data were analyzed by FIJI image process software (see details in Materials and Methods). The fluorescence intensity in the photobleached area along with the time was plotted using GraphPad Software Prism.
E: VPS8D knockdown cell expressing Dop1p-mNeon analyzed by FRAP. Three images were extracted from a video (Movie 2) showing a photobleaching event and recovery, with panels as in Fig. 2C but with the bleaching event at t=0:26 and recovery at t=5:33.
F: Recovery after photobleaching, plotted as in Fig. 2D.
G: Dop1p recovery after photobleaching is slowed in VPS8Dknockdown cell. Three FRAP experiments obtained from wild type andVPS8D knockdown cells were analyzed and calculated to obtain the mean±s.d. recovery rate after photobleaching. The data were plotted using GraphPad Software Prism and using a two-tailed t -test. The difference between wild type and VPS8D knockdown is significant,P -value <0.05.
H: Contractile cycle period is increased in VPS8D knockdown cells. The contractile cycle period of wild type cell and VPS8Dknockdown cell were measured. 50 cycles from wild type cells and 22 cycles from VPS8D knockdown cells were measured to obtain the contractile cycle period and the data were plotted using GraphPad Software Prism and using a two-tailed t -test. The difference between wild type and VPS8D knockdown is significant,P -value <0.0001.
I: Vma4p localization in WT and VPS8D knockdown cells. Growing cells expressing Vma4p-mNeon were imaged to show Vma4p localization in WT (left panel) and VPS8D knockdown cells (right panel). Images were taken with a Zeiss Axio Observer 7 system.
J: The spongiome area is reduced in VPS8D knockdown cell.VPS8D -KD growing cells expressing Vma4p-mNeon were treated with cadmium (+Cd) for 3,5 and 8 hours or without cadmium (-Cd) and then were fixed for imaging. 20 images for each sample were analyzed to quantify the area of the Vma4p-labelled spongiome and the whole cell (Fig. S3). The data were plotted using GraphPad Software Prism and using a two-tailed t -test. The ratio of the mean±s.d. volume of spongiome to whole cell from each sample is shown. The difference between +Cd and -Cd at 5- and 8-hours treatment point are significant, P -value <0.0001.
Figure 3: