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Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM

View Article: PubMed Central - PubMed

ABSTRACT

Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent protein-labeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.

No MeSH data available.


Interaction between wild-type and mutant affects localization of individual forms of NPM.(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).
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pone.0175175.g003: Interaction between wild-type and mutant affects localization of individual forms of NPM.(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).

Mentions: We have previously checked the tug-of-war hypothesis described by Bolli et al [27] suggesting that the localization of both fluorescently labeled wt and mutated NPM forms depends on their mutual ratio. We showed that the abundance of one NPM form caused partial redistribution of its oligomer partner in Hela cells co-transfected with eGFP_NPMmutA and mRFP1_NPMwt [26]. Here we analyzed the distribution of fluorescently labeled NPM variants in HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA or eGFP_NPMmutE (Fig 3a). For both mutation types, co-transfection with wt caused significant changes in the subcellular distribution: higher fraction of eGFP_NPMmut in the nucleolus as well as a fraction of mRFP1_NPMwt in the cytoplasm was observed in comparison with the distribution in single form-transfected cells (Fig 3b and 3c, S1 and S2 Tables). Our observations prove the fact that the ability of NPM to form oligomers is not disrupted by any type of C-terminal mutation and that heterooligomers between the wild-type and mutated NPM are formed affecting the localization of each other.


Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM
Interaction between wild-type and mutant affects localization of individual forms of NPM.(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).
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pone.0175175.g003: Interaction between wild-type and mutant affects localization of individual forms of NPM.(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).
Mentions: We have previously checked the tug-of-war hypothesis described by Bolli et al [27] suggesting that the localization of both fluorescently labeled wt and mutated NPM forms depends on their mutual ratio. We showed that the abundance of one NPM form caused partial redistribution of its oligomer partner in Hela cells co-transfected with eGFP_NPMmutA and mRFP1_NPMwt [26]. Here we analyzed the distribution of fluorescently labeled NPM variants in HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA or eGFP_NPMmutE (Fig 3a). For both mutation types, co-transfection with wt caused significant changes in the subcellular distribution: higher fraction of eGFP_NPMmut in the nucleolus as well as a fraction of mRFP1_NPMwt in the cytoplasm was observed in comparison with the distribution in single form-transfected cells (Fig 3b and 3c, S1 and S2 Tables). Our observations prove the fact that the ability of NPM to form oligomers is not disrupted by any type of C-terminal mutation and that heterooligomers between the wild-type and mutated NPM are formed affecting the localization of each other.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent protein-labeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.

No MeSH data available.