LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

4.3.9 De novo desmosome formation is impaired in 14-3-3γ down regulated cells.
To determine whether 14-3-3γ is required for incorporation of desmosomal components
during desmosome biogenesis, a calcium switch assay was performed as previously
described (19). The HCT116-derived 14-3-3γ knockdown clone and the vector control
were grown in low calcium medium for 20 hrs and then shifted to medium with standard
calcium levels. The cells were fixed for immunofluorescence experiments at 30 min and
60 min time intervals and stained with antibodies to DSC2/3, PG, and E-cadherin. As
previously reported by us (122), PG is present at the cell border in the absence of calcium
and the levels at the border increase upon calcium addition. A diffused cytoplasmic
staining was observed for DSC2/3 in cells cultured in calcium depleted conditions
(Figure 4.3.19 A and B). These proteins gradually accumulated at the cell-cell border and
showed typical membrane localization after 60min (Figure 4.3.19 A and B upper panels).
However these proteins failed to localize to the cell border in the 14-3-3γ knockdown
clone upon addition of calcium (Figure 4.3.19 (A and B)). The localization of E- cadherin
at the cell border was not altered in the vector as well as in 14-3-3γ knockdown cells in
the presence or absence of calcium (Figure 4.3.19 C). However, E-cadherin showed an
increase in the cell border intensity in both vector control and 14-3-3γ knockdown clone
when shifted from a low calcium medium to standard calcium medium, which is
consistent with previously reported experiments from our laboratory (122). These results
suggest that 14-3-3γ is required for the localization of the desmosomal components to the
cell border during desmosome formation and is not required for the assembly of adherens
junctions.
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