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Dissecting the cell to nucleus, perinucleus and cytosol.

Shaiken TE, Opekun AR - Sci Rep (2014)

Bottom Line: The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei.Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure.The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
Cells have been described under the microscope as organelles containing cytoplasm and the nucleus. However, an unnoted structure exists between the cytoplasm and the nucleoplasm of eukaryotic cells. In addition to the nuclear envelope, there exists a perinuclear region (PNR or perinucleus) with unknown composition and function. Until now, an investigation of the role of the perinucleus has been restricted by the absence of a PNR isolation method. This manuscript describes a perinucleus isolation technique on the basis of its unique compact organization. The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei. Using four different normal and cancer cell lines, it was shown that the composition of PNR is highly dynamic. Application of the method showed that translocation of the p53 tumor-suppressor protein to the perinucleus in immortalized MEF cells is correlated with the translocation of p53-stabilizing protein, nucleophosmin (B23), to the PNR. Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure. The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.

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Phase contrast images of MDA-MB-435 cells and isolated nuclei.(A) MDA-MB-435 cells. (B) Nuclei isolated in isotonic buffer A. Cytoplasm of the cell at this stage was removed by detergent-containing buffer; approximately 65% of proteins were extracted to the cytosol; nuclei contain perinuclear region proteins; nucleoli are visible. (C) Nuclei after extraction of the perinuclear region with buffer B. The core nucleus does not collapse after removal of the perinuclear region proteins; approximately 20% of total cellular proteins were extracted with the perinuclear fractionation; nucleoli are visible. (D) Nuclei isolated with the classical method of using hypotonic buffer. The shape of nuclei varies; some “fibrous” structures around nuclei are visible; nuclei contain nucleoli.
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f1: Phase contrast images of MDA-MB-435 cells and isolated nuclei.(A) MDA-MB-435 cells. (B) Nuclei isolated in isotonic buffer A. Cytoplasm of the cell at this stage was removed by detergent-containing buffer; approximately 65% of proteins were extracted to the cytosol; nuclei contain perinuclear region proteins; nucleoli are visible. (C) Nuclei after extraction of the perinuclear region with buffer B. The core nucleus does not collapse after removal of the perinuclear region proteins; approximately 20% of total cellular proteins were extracted with the perinuclear fractionation; nucleoli are visible. (D) Nuclei isolated with the classical method of using hypotonic buffer. The shape of nuclei varies; some “fibrous” structures around nuclei are visible; nuclei contain nucleoli.

Mentions: In order to isolate the perinuclear region, the melanoma-derived MDA-MB-435 cell line was used as a model9 (Figure 1A). MDA-MB-435 cells were lysed with Buffer A and the nuclei were sequentially washed with low- and no-detergent containing buffer A (Figure 1B). The perinuclear proteins were extracted with buffer B (Figure 1C) and the core nuclear fraction (cNF) was dissolved in 8 M urea. The nuclei also were isolated with the classical scheme of fractionation in hypotonic buffer10, which was used as a control (Figure 1D). As a control for the fractionation, Chaps-containing buffer11 was applied for cell lysis. Buffer A resulted in extraction of approximately 70% of the cytoplasmic protein of the cell (Table 1). Further fractionation of nuclei (shown in Figure 1B) extracted approximately 15–18% more proteins, which are believed to compose the compact perinuclear (PNF) fraction (Table 1). Cancer cell lines MDA-MB-435 and HeLa and immortalized MEF cells contained higher concentrations of protein in the PNF than primary MEF cells. After PNF extraction, it was observed that the nuclei did not collapse and retained the nucleoli (Figure 1C), accounting for approximately 15% of the total cellular protein (Table 1). Total nuclear proteins of the perinuclear fraction and core nuclear fractions (PNF + cNF) compose approximately 30% of total cellular proteins.


Dissecting the cell to nucleus, perinucleus and cytosol.

Shaiken TE, Opekun AR - Sci Rep (2014)

Phase contrast images of MDA-MB-435 cells and isolated nuclei.(A) MDA-MB-435 cells. (B) Nuclei isolated in isotonic buffer A. Cytoplasm of the cell at this stage was removed by detergent-containing buffer; approximately 65% of proteins were extracted to the cytosol; nuclei contain perinuclear region proteins; nucleoli are visible. (C) Nuclei after extraction of the perinuclear region with buffer B. The core nucleus does not collapse after removal of the perinuclear region proteins; approximately 20% of total cellular proteins were extracted with the perinuclear fractionation; nucleoli are visible. (D) Nuclei isolated with the classical method of using hypotonic buffer. The shape of nuclei varies; some “fibrous” structures around nuclei are visible; nuclei contain nucleoli.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4017230&req=5

f1: Phase contrast images of MDA-MB-435 cells and isolated nuclei.(A) MDA-MB-435 cells. (B) Nuclei isolated in isotonic buffer A. Cytoplasm of the cell at this stage was removed by detergent-containing buffer; approximately 65% of proteins were extracted to the cytosol; nuclei contain perinuclear region proteins; nucleoli are visible. (C) Nuclei after extraction of the perinuclear region with buffer B. The core nucleus does not collapse after removal of the perinuclear region proteins; approximately 20% of total cellular proteins were extracted with the perinuclear fractionation; nucleoli are visible. (D) Nuclei isolated with the classical method of using hypotonic buffer. The shape of nuclei varies; some “fibrous” structures around nuclei are visible; nuclei contain nucleoli.
Mentions: In order to isolate the perinuclear region, the melanoma-derived MDA-MB-435 cell line was used as a model9 (Figure 1A). MDA-MB-435 cells were lysed with Buffer A and the nuclei were sequentially washed with low- and no-detergent containing buffer A (Figure 1B). The perinuclear proteins were extracted with buffer B (Figure 1C) and the core nuclear fraction (cNF) was dissolved in 8 M urea. The nuclei also were isolated with the classical scheme of fractionation in hypotonic buffer10, which was used as a control (Figure 1D). As a control for the fractionation, Chaps-containing buffer11 was applied for cell lysis. Buffer A resulted in extraction of approximately 70% of the cytoplasmic protein of the cell (Table 1). Further fractionation of nuclei (shown in Figure 1B) extracted approximately 15–18% more proteins, which are believed to compose the compact perinuclear (PNF) fraction (Table 1). Cancer cell lines MDA-MB-435 and HeLa and immortalized MEF cells contained higher concentrations of protein in the PNF than primary MEF cells. After PNF extraction, it was observed that the nuclei did not collapse and retained the nucleoli (Figure 1C), accounting for approximately 15% of the total cellular protein (Table 1). Total nuclear proteins of the perinuclear fraction and core nuclear fractions (PNF + cNF) compose approximately 30% of total cellular proteins.

Bottom Line: The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei.Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure.The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
Cells have been described under the microscope as organelles containing cytoplasm and the nucleus. However, an unnoted structure exists between the cytoplasm and the nucleoplasm of eukaryotic cells. In addition to the nuclear envelope, there exists a perinuclear region (PNR or perinucleus) with unknown composition and function. Until now, an investigation of the role of the perinucleus has been restricted by the absence of a PNR isolation method. This manuscript describes a perinucleus isolation technique on the basis of its unique compact organization. The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei. Using four different normal and cancer cell lines, it was shown that the composition of PNR is highly dynamic. Application of the method showed that translocation of the p53 tumor-suppressor protein to the perinucleus in immortalized MEF cells is correlated with the translocation of p53-stabilizing protein, nucleophosmin (B23), to the PNR. Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure. The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.

Show MeSH
Related in: MedlinePlus