Fig 1.
Schematic representation of p53 protein domains with indicated positions of the NES and NLS sequences.
The amino acids mutated in this study are in red and marked with an arrow.
Fig 2.
Cellular localization of mRFP1-labeled variants of p53 (R_p53) co-expressed with Cerulean labeled NPMmut (C_NPMmut).
A) p53wt, B) monomeric variants, and C) putative dimeric variants. Bar represents 20 µm.
Fig 3.
Immunoblot of GA-crosslinked lysates from HEK-293T cells transfected with mVenus-labeled p53 variants.
Positions of monomers, dimers and tetramers are marked with arrows.
Fig 4.
A) Intensity (upper two rows of the respective panel) and FLIM (bottom row of the respective panel) images of p53 variants in HEK-293T cells co-transfected with two different color variants of the specific FP-tagged protein (V_p53 donor/R_p53 acceptor).
Left columns (start): initial state, right columns (bleach): images after photodestruction of mRFP1 in cells marked with the dotted line by 561 nm light. The numbers refer to fluorescence lifetimes in the corresponding cells (in ns). Bar 20 µm. B) Statistical evaluation of the fluorescence lifetime changes in the heteroFRET experiments. Mean values are plotted with ±SD (left), red symbols in the 95% confidence graph (right) mark significant differences between appropriate pairs of samples (p < 0.05).
Fig 5.
FCCS measurements in the nucleus of HEK-293T cells co-transfected with a mix of mVenus- and mRFP1-labeled p53 variants.
A) The cross-correlation amount Q was determined for each FCCS measurement. B) The diffusion time τD was calculated from autocorrelation curves recorded in the mVenus detection channel. Mean values are plotted with ±SD (left), red symbols in the 95% confidence graph (right) mark significant differences between appropriate pairs of samples (p < 0.05).
Fig 6.
A) Subcellular localization of p53 mutants tagged with nowGFP in HEK-293T cells.
B) Immunoblot of nowGFP-labeled I332S using GA-crosslinked lysates from HEK-293T cells, Comparison with p53wt and other monomeric variants, C) Comparison of fluorescence anisotropy changes. Mean values are plotted with ±SD, red symbols in the 95% confidence graph (right) mark significant differences between appropriate pairs of samples (p < 0.05)., D) Localization of nowGFP-labeled I332S in HEK-293T cells. Bar represents 20 µm.
Fig 7.
Characteristics of mRFP1-labeled p53 variants with impaired NLS expressed in HEK-293T cells.
A) Immunoblot of GA-crosslinked lysates with p53 variants detected by anti-RFP (left) or anti-p53 (right) antibody. B) Representative images of the subcellular localization. Bar represents 20 µm.
Fig 8.
Co-immunoprecipitation of NPM and p53 variants.
A) Statistical analysis of p53 immunoblot band intensities co-precipitated with NPMmut relative to the p53wt (mean ± SD from at least 3 independent experiments). B) Representative immunoblot of NPMmut and p53 levels in the total cell lysate (Input) and NPMmut-precipitate (IP: C_NPMmut). C) Representative immunoblot of NPMwt and p53 levels in the total cell lysate (Input) and NPMwt-precipitate (IP: C_NPMwt).
Fig 9.
Stability analysis of mRFP1_p53 variants.
Transfected HEK-293T cells were lysed under native conditions and seminative PAGE with varying SDS concentration was performed. Detection of p53 (upper row) and RFP (lower row) revealed different SDS thresholds for decomposition of p53 complexes.
Table 1.
Pathogenicity score of different p53 point mutations according to AlphaFold2 [93].