Journal of Cell Science Accepted manuscriptjcs.biologists.org/content/joces/early/2014/03/07/jcs.125807.full.pdf · 1 14-3-3γ meditated transport of plakoglobin to the cell border
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14-3-3γ meditated transport of plakoglobin to the cell border is required for the initiation
Transduction laboratories, dilution 1:100) were incubated with the cells for 1 hour at
room temperature at the indicated dilutions as described (Gosavi et al., 2011). To stain
mitochondria, Mitotraker GreenFM (Invitrogen) was used at 100nM to stain live cells.
Confocal images were obtained by using a LSM 510 Meta Carl Zeiss confocal system
with an Argon 488 nm and Helium/Neon 543 nm lasers. All images were obtained using
an Axio Observer Z.1 microscope (numerical aperture [NA] 1.4) at a magnification of
630X with 2X or 4X optical zoom. The surface intensity of staining was measured for the
different proteins in a minimum of twenty four cells using the Axiovision software and
the mean and standard deviation plotted.
Hanging drop assays. Hanging drop assays were used to measure cell adhesion as
previously described (Kundu et al., 2008).
GST pulldown and immunoprecipitation assays. These assays were performed as
previously described (Dalal et al., 1999).
GST PG production and kinase assays. GST alone or GST PG1-300 was purified from
bacteria as described (Dalal et al., 2004). Purified proteins were used in kinase assays
using recombinant PKCµ and a peptide derived from CREB as a positive control (Signal
Chem) and kinase activity determined using the ADP glow assay kit (Promega) according
to the manufacturer’s instructions.
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Histology and Immunohistochemistry. Mouse testes were fixed in 10% formaldehyde
overnight and processed for histology as described (Kundu et al., 2008). TUNEL assays
were performed as per the manufacturer’s instructions (Promega).
Electron microscopy. WT and 14-3-3γ KNOCKDOWN testis were fixed with 3%
glutaraldehyde and postfixed with 1% osmium tetraoxide (Tedpella). Grids were
contrasted with alcoholic uranyl acetate for 1 minute and lead citrate for half a minute.
The grids were observed under a Carl Zeiss LIBRA120 EFTEM transmission electron
microscope at an accelerating voltage of 120 kV and at 325,000 magnification. Images
were captured using a Slow Scan CCD camera (TRS, Germany).
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Acknowledgements. We thank Dr.’s Young Lee, Stefan Linder, Tetsu Akiyoma, Chen
Gu and Lawrence Goldstein for supplying us with constructs used during the course of
this study. We would also like to thank the ACTREC imaging facility for help with
confocal microscopy, the ACTREC animal facility and Ms. Sharda. Sawant for helping
with the preparation of grids for electron microscopy.
This work was supported by grants from the Department of Biotechnology
(http://dbtindia.nic.in/index.asp) (grants BT/PR6521/Med/14/828/2005 and
BT/PR12578/MED/31/75/2009) and ACTREC (www.actrec.gov.in) (LS and SND) and a
fellowship from the University Grants Commission (http://www.ugc.ac.in/) (AM).
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Figure Legends.
Figure 1. Loss of 14-3-3γ leads to disruption of cell-cell adhesion. A-D. Tissue
sections from mouse testis injected with either the 14-3-3γ knockdown construct (sh14-3-
3γ) or the vector control (Vec) were stained with antibodies to 14-3-3γ and visualized by
light microscopy (A and D) or stained with hematoxylin and eosin to visualize either the
seminiferous tubules (top panels) or the epididymis (bottom panels) (B). The percentage
of vesicles containing spermatozoa in the epididymis is graphed on the y-axis and the bar
represents the mean and standard deviation for three different animals (C). Bars in A
correspond to 5µM, B to 5µM and D to 5µM. E. Electron micrographs of the testis
injected with either the 14-3-3γ KNOCKDOWN viruses (sh14-3-3γ) or the vector control
(Vec). Sertoli cells (SC) and germ cells (GC) are indicated. The panels on the extreme
right are higher magnification images of the boxed areas as indicated. Bars correspond to
2µM.
Figure 2. Loss of 14-3-3γ leads to a decrease in cell-cell adhesion. A. Protein extracts
from the Vec and sh14-3-3γ cells were resolved on SDS-PAGE gels followed by Western
blotting with the indicated antibodies. B. mRNA was prepared from the Vec and sh14-3-
3γ cells and RT-PCR reactions performed with oligonucleotide pairs specific for the
indicated genes. GAPDH served as a loading control. C-D. Hanging drop assays were
performed on the Vec and sh14-3-3γ cells. The images of the clumps (C) and the
quantitation (D) of cluster number and size are shown. Bars in C correspond to 200µM.
E. Protein extracts from the Vec and sh14-3-3ε cells were resolved on SDS-PAGE gels
followed by Western blotting with the indicated antibodies. F-G. Hanging drop assays
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were performed on the Vec and sh14-3-3γ cells. The images of the clumps (F) and the
quantitation (G) of cluster number and size are shown. Bars in F correspond to 200µM.
Figure 3. Localization of PG is altered in the sh-14-3-3γ cells. A-B. Protein extracts
from the Vec and sh14-3-3γ cells were resolved on SDS-PAGE gels followed by Western
blotting with the indicated antibodies. C. mRNA was prepared from the Vec and sh14-3-
3γ cells and RT-PCR reactions performed with oligonucleotide pairs specific for the
indicated genes. GAPDH served as a loading control. D. PG levels at the border were
determined in the vector control cells (Vec) and the 14-3-3ε knockdown cells (sh14-3-3ε)
Bars correspond to 5µM. Note that PG levels did not change at the border upon
knockdown of 14-3-3ε. E-F. Vec and sh14-3-3γ cells were stained with the indicated
antibodies followed by confocal microscopy The bars in E correspond to 5µM and the
bars in F correspond to 10µM. Representative images are shown. The border intensity
was measured for at least twenty cells in three different experiments. The mean and
standard deviation for three independent experiments is shown.
Figure 4. 14-3-3γ is required to initiate desmosome formation. A. sh14-3-3γ cells
were transfected with either GFP alone or GFP 14-3-3γR. The cells were stained with
antibodies to PG (red), followed by confocal microscopy. Bars correspond to 5µM. B.
Protein extracts from HCT116 cells were incubated with either GST or GST14-3-3γ. The
reactions were resolved on SDS-PAGE gels followed by Western blotting with the
indicated antibodies. C. Vec and sh14-3-3γ cells were incubated in low calcium medium
for 24 hours (0 minute). After calcium addition for 60 min, cells were fixed and stained
with the indicated antibodies followed by confocal microscopy. Note that the levels of the
desmosomal proteins do not increase at the border in the sh-14-3-3γ cells as compared to
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the Vec cells. No change in E-cadherin staining is observed. Bars correspond to 10 µM.
The border intensity was measured for at least 20 cells in three different experiments. The
mean and standard deviation for three independent experiments is shown.
Figure 5. 14-3-3γ association with PG requires PKCµ activity. A and B. HCT116
cells were transfected with either MYC-epitope tagged WT PG or the S236A mutant of
PG. 48 hours post transfection protein extracts were incubated with GST or GST14-3-3γ
followed by Western blotting with antibodies to MYC (A) or the cells were stained with
antibodies to the MYC-epitope (B). DIC images for the fields are shown in the lower
panels. Bars correspond to 10µM. C and D. HCT116 cells were treated with either the
vehicle control (DMSO), the pan PKC inhibitor (BisI) and the PKCα and PKCµ specific
inhibitor (Go6976). Protein extracts from these cells were incubated with either GST or
GST14-3-3γ, the reactions resolved on SDS-PAGE gels and Western blots performed
with the indicated antibodies (C) or stained with antibodies to PG (D). Bars correspond to
5µM. E-F. Protein extracts from vector control or PKCµ knockdown cells (sh PKCµ)
were resolved on SDS-PAGE gels and Western blots performed with the indicated
antibodies. Note that there is a decrease in PG and DP levels upon PKCµ knockdown but
no difference in PKP3 levels is observed. F. Vec cells and sh PKCµ cells were fixed and
stained with the indicated antibodies. Bars correspond to 5µM. G. PG 1-300 was
produced in bacteria as a GST fusion and kinase assays performed using recombinant
PKCµ according to the manufacturer’s instructions. GST alone was used as a negative
control in this assay. Different concentrations of the substrate are shown on the x-axis
and enzyme activity is graphed on the y-axis and bars represent standard deviation. Note
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that an increase in kinase activity is observed upon an increase in the concentration of
PG1-300 but not with an increase in the concentration of GST alone.
Figure 6. KIF5B is required for transport of PG to the cell border. A. HCT116 cells
were transfected with the vector control (pCDNA3) or HA-14-3-3γ and
immunoprecipitations were performed with antibodies to the HA epitope. The reactions
were resolved on SDS-PAGE gels and Western blots performed with the indicated
antibodies. B. HCT116 cells were transfected with constructs expressing an shRNA
targeting KIF5B. Individual cell clones were expanded and protein extracts from these
clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to
KIF5B. Note that the knockdown clones have a lower level of KIF5B (K1-K5) than the
vector control (Vec). A Western blot for actin served as a loading control. C. Vector
control (Vec) or KIF5B knockdown clones (K3 and K5) were incubated with Mitotraker
GreenFM (Invitrogen). Bars correspond to 10µM. D. Hanging drop assays were
performed to determine cell-cell adhesion. K3 and K5 formed fewer and smaller clumps
as compared to the Vec cells. E. Vec, K3 and K5 cells were stained with antibodies to
PG, DSC2/3, DSG2, DP, and PKP3 and surface intensity quantitated using confocal
microscopy. Total magnification was 630X with 2X optical zoom. Bars correspond to
5µM. F. Protein extracts prepared from the Vec, K3 and K5 cells were resolved on SDS-
PAGE gels followed by Western blotting with the indicated antibodies. G. Vec, K3 and
K5 cells were stained with antibodies to p120 catenin and α-Ecatenin and surface
intensity quantitated using confocal microscopy. Total magnification is 630X with 2X
optical zoom. Bars correspond to 5µM.
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Figure 7. Dominant negative mutants of KIF5B and KLC1 inhibit PG transport to
the border. A. HCT116 cells were transfected with YFP-tagged WT or DN KIF5B or
GFP-tagged WT or DN KIF3A. Post transfection the cells were fixed and stained with
antibodies to PG. B. Protein extracts from HCT116 cells were incubated with GST alone
or GST-KLC1 or GST-KLC2. The reactions were resolved on SDS-PAGE gels followed
by Western blotting with the indicated antibodies. C. Cartoon of KLC1 mutants. D.
Protein extracts from HCT116 cells were incubated with GST alone or GST-KLC1WT or
GST-KLC1 TPR and GST-KLC1 CC. The reactions were resolved on SDS-PAGE gels
followed by Western blotting with the indicated antibodies. E. GFP-KLC1 or GFP-KLC1
CC were transfected into HCT116 cells. Forty-eight hours post transfection the cells were
fixed and stained with antibodies to PG. DNA was stained with DAPI. Total
magnification is 630X with 2X optical zoom. All bars correspond to 5µM.
Figure 8. Loss of KIF5B and 14-3-3γ in the testis leads to a disruption of desmosome
formation and sterility. A-D. Lentiviruses encoding either the vector control or
shRNA’s targeting either 14-3-3ε, 14-3-3γ or KIF5B were injected into the testes of
Swiss mice. Thirty-five days post-injection the mice were sacrificed, sections of the
epididymis and testes were stained with hematoxylin and eosin and examined
microscopically (A). A immunohistochemical analysis with antibodies to the different
proteins that were knocked down demonstrated that the expression of 14-3-3ε, 14-3-3γ
and KIF5B were inhibited in the testis injected with the appropriate lentivirus (B). Bar
corresponds to 20µM. The percentage of epididymal vesicles showing the presence of
mature spermatozoa in three different animals was determined and the mean and standard
deviation plotted (C). Testis sections were stained with antibodies to PG, PKP3, DSC2/3,
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N-cadherin and E-cadherin Bar corresponds to 10µM. (D). Total magnification was