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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.

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Subcellular distribution of mutated NPM depends on mutation type.(a) eGFP fluorescence from HEK-293T cells transfected with eGFP plasmid (GFP), eGFP_NPMwt (wt), eGFP_NPMmutA (mutA), eGFP_NPMmutB (mutB) or eGFP_NPMmutE (mutE) showing various subcellular distribution of individual NPM variants. The bars represent 20μm. (b) fraction of transfected cells displaying eGFP_NPMmutA (or E) signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bars). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between mutA and mutE obtained from two-way ANOVA test was P < 0.001 (***). (c) immunoblot of lysates from HEK-293T cells transfected with individual NPM variants. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Densitometric evaluation of NPM exo/endo level and the ratio of NPMexo/endo expression vs the transfection efficiency (20%, 15%, 13,9% resp. 17,8% for wt, mutA, mutB resp. mutE) are indicated for the individual cell lines.
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pone.0175175.g002: Subcellular distribution of mutated NPM depends on mutation type.(a) eGFP fluorescence from HEK-293T cells transfected with eGFP plasmid (GFP), eGFP_NPMwt (wt), eGFP_NPMmutA (mutA), eGFP_NPMmutB (mutB) or eGFP_NPMmutE (mutE) showing various subcellular distribution of individual NPM variants. The bars represent 20μm. (b) fraction of transfected cells displaying eGFP_NPMmutA (or E) signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bars). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between mutA and mutE obtained from two-way ANOVA test was P < 0.001 (***). (c) immunoblot of lysates from HEK-293T cells transfected with individual NPM variants. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Densitometric evaluation of NPM exo/endo level and the ratio of NPMexo/endo expression vs the transfection efficiency (20%, 15%, 13,9% resp. 17,8% for wt, mutA, mutB resp. mutE) are indicated for the individual cell lines.

Mentions: We transfected HEK-293T cell line with eGFP-labeled variants of NPM and examined the eGFP_NPM subcellular localization under the confocal microscope (Fig 2a). The wild-type NPM and three types of mutated NPM (A, B and E) were analyzed. While the eGFP_NPMwt was detected solely in nucleoli, more than 80% of eGFP_NPMmutA-transfected cells exhibited exclusively cytoplasmic localization of the mutated protein (Fig 2b). A combination of eGFP_NPMmutA signal from the nucleolus with cytoplasmic staining was observed in approximately 15% of the transfected cells. Moreover, the relative fluorescence intensity from the remaining cells, showing eGFP_NPMmutA signal only in the nucleoli (approximately 5% of transfected cells), was weak, indicating low plasmid amplification in these cells. Subcellular distribution of eGFP signal in cells transfected with eGFP_NPMmutB was almost identical as for NPMmutA (Fig 2a). On the contrary, 65% of cells transfected with eGFP_NPMmutE displayed eGFP fluorescence from the nucleolus, whether exclusively or partially (i.e. signal was detected from both the cytoplasm and the nucleoli) (Fig 2a and 2b, S1 Table). Identical results were obtained with plasmids containing the red form of the fluorescence protein, mRFP1, instead of eGFP (data not shown). The transfection efficiency measured by flow cytometry was about 45% in all samples and high expression of recombinant fusion proteins was confirmed by immunoblot (Fig 2c, S1 Fig).


Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM
Subcellular distribution of mutated NPM depends on mutation type.(a) eGFP fluorescence from HEK-293T cells transfected with eGFP plasmid (GFP), eGFP_NPMwt (wt), eGFP_NPMmutA (mutA), eGFP_NPMmutB (mutB) or eGFP_NPMmutE (mutE) showing various subcellular distribution of individual NPM variants. The bars represent 20μm. (b) fraction of transfected cells displaying eGFP_NPMmutA (or E) signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bars). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between mutA and mutE obtained from two-way ANOVA test was P < 0.001 (***). (c) immunoblot of lysates from HEK-293T cells transfected with individual NPM variants. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Densitometric evaluation of NPM exo/endo level and the ratio of NPMexo/endo expression vs the transfection efficiency (20%, 15%, 13,9% resp. 17,8% for wt, mutA, mutB resp. mutE) are indicated for the individual cell lines.
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pone.0175175.g002: Subcellular distribution of mutated NPM depends on mutation type.(a) eGFP fluorescence from HEK-293T cells transfected with eGFP plasmid (GFP), eGFP_NPMwt (wt), eGFP_NPMmutA (mutA), eGFP_NPMmutB (mutB) or eGFP_NPMmutE (mutE) showing various subcellular distribution of individual NPM variants. The bars represent 20μm. (b) fraction of transfected cells displaying eGFP_NPMmutA (or E) signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bars). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between mutA and mutE obtained from two-way ANOVA test was P < 0.001 (***). (c) immunoblot of lysates from HEK-293T cells transfected with individual NPM variants. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Densitometric evaluation of NPM exo/endo level and the ratio of NPMexo/endo expression vs the transfection efficiency (20%, 15%, 13,9% resp. 17,8% for wt, mutA, mutB resp. mutE) are indicated for the individual cell lines.
Mentions: We transfected HEK-293T cell line with eGFP-labeled variants of NPM and examined the eGFP_NPM subcellular localization under the confocal microscope (Fig 2a). The wild-type NPM and three types of mutated NPM (A, B and E) were analyzed. While the eGFP_NPMwt was detected solely in nucleoli, more than 80% of eGFP_NPMmutA-transfected cells exhibited exclusively cytoplasmic localization of the mutated protein (Fig 2b). A combination of eGFP_NPMmutA signal from the nucleolus with cytoplasmic staining was observed in approximately 15% of the transfected cells. Moreover, the relative fluorescence intensity from the remaining cells, showing eGFP_NPMmutA signal only in the nucleoli (approximately 5% of transfected cells), was weak, indicating low plasmid amplification in these cells. Subcellular distribution of eGFP signal in cells transfected with eGFP_NPMmutB was almost identical as for NPMmutA (Fig 2a). On the contrary, 65% of cells transfected with eGFP_NPMmutE displayed eGFP fluorescence from the nucleolus, whether exclusively or partially (i.e. signal was detected from both the cytoplasm and the nucleoli) (Fig 2a and 2b, S1 Table). Identical results were obtained with plasmids containing the red form of the fluorescence protein, mRFP1, instead of eGFP (data not shown). The transfection efficiency measured by flow cytometry was about 45% in all samples and high expression of recombinant fusion proteins was confirmed by immunoblot (Fig 2c, S1 Fig).

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.


Related in: MedlinePlus