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: