VPS8D plays an important role in cellular resistance to osmotic stress.
A, left: CVC anatomy in T. thermophila . The cartoon shows previously established features of the Tetrahymena CVC including one central bladder near the cell posterior, the surrounding tubulo-vesicular spongiome network, and two pores connecting the bladder to the plasma membrane. Dop1p (blue) primarily labels the bladder, and Vma4p (green) exclusively labels the spongiome. Also shown are the oral apparatus (OA) in the anterior end of cell, and both the micronucleus (MIC) and macronucleus (MAC) near the cell midpoint. The vertical rows represent the cytoskeletal tracks called primary meridians, along which the ciliary basal bodies are spaced. For clarity, the cilia themselves are only shown at the cell edges.
A, right: As previously reported, Vps8Dp localizes to the CVC as well as to cytosolic puncta. Growing cells expressing Vps8Dp endogenously tagged with mCherry were fixed and stained with DAPI. Images were taken with a Marianas spinning disc confocal microscope.
B: VPS8D RNAi knockdown strategy via hairpin expression. Cell lines transformed with the hairpin construct, under the control of the inducible MTT1 promoter, are called VPS8D -KD.
C: VPS8D expression is reduced in cells expressing theVPS8D hairpin. The accumulation of Vps8Dp-FLAG, in cells expressing the tagged protein from the endogenous locus, was measured by anti-FLAG immunostaining. The labels “-Cd” and “+Cd” indicate the presence or absence of cadmium, which induces VPS8D hairpin expression. Images were taken with a Zeiss Axio Observer 7 system. Each image was generated from10 z-sections across the CVC using the Z project tool in FIJI software.
D: Quantification of Vps8Dp-FLAG accumulation without/with hairpin-induced knockdown. 10 images for each condition (+Cd and -Cd) were used to quantify the intensity of CVC-localized Vps8Dp immunofluorescence. The data were plotted to a box-and-whiskers plot with statistical analysis via two-tailed t -test by GraphPad Software Prism. The boxes display median and interquartile ranges, and the whiskers represent the minimum to maximum value of data. The difference between +Cd and -Cd is significant, P -value <0.001.
E: VPS8D knockdown (VPS8D -KD) cells fail to restore normal cell shape after hypoosmotic-induced swelling. DIC images from wild type (WT) and VPS8D -KD cells are shown. Images were taken with a Zeiss Axio Observer 7 system.
F: VPS8D knockdown cells do not survive hypoosmotic stress. Wild type (WT) and VPS8D -KD cells were incubated with (+Cd) or without cadmium (-Cd), and then given a hypoosmotic challenge. After 16h, cell viability was measured (see details in Materials and Methods). The data were plotted using GraphPad Software Prism, using a two-tailed t -test. Individual data points and the mean±s.d. percentage of survival are shown. The P -value for the difference between WT and VPS8D -KD is <0.0001.
G: Differences in the responses after hypoosmotic shock between wild type and VPS8D -KD cells.
Figure 2: