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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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Patterns of protein distribution in MDA-MB-435 cells.Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for CDS method; Nuc. Cont is a control for nuclear proteins obtained with classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from cytoplasm. They are not detected in perinuclear and nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques (B) Proteins detected in the cytosol and the perinuclear fraction: proteins were detected as cytosolic proteins with both cellular lysis technique; in addition, these proteins also appeared in the perinuclear fraction by extraction with Buffer B. (C) Proteins of perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. (D) Proteins detected in nuclear and perinuclear fractions: transcription factors (that supposedly belong to the nuclear fraction) also appeared in the perinuclear fraction. (E) Nuclear proteins: proteins were detected in the nuclear fraction. Nuclear proteins were obtained with both new and classical nuclei isolation techniques. The PVDF membranes were cropped into two halves and the high and low molecular weight proteins were shown correspondingly.
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f2: Patterns of protein distribution in MDA-MB-435 cells.Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for CDS method; Nuc. Cont is a control for nuclear proteins obtained with classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from cytoplasm. They are not detected in perinuclear and nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques (B) Proteins detected in the cytosol and the perinuclear fraction: proteins were detected as cytosolic proteins with both cellular lysis technique; in addition, these proteins also appeared in the perinuclear fraction by extraction with Buffer B. (C) Proteins of perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. (D) Proteins detected in nuclear and perinuclear fractions: transcription factors (that supposedly belong to the nuclear fraction) also appeared in the perinuclear fraction. (E) Nuclear proteins: proteins were detected in the nuclear fraction. Nuclear proteins were obtained with both new and classical nuclei isolation techniques. The PVDF membranes were cropped into two halves and the high and low molecular weight proteins were shown correspondingly.

Mentions: A slight difference was visible under the phase contrast microscope between nuclei fractionated with the different nuclear-extraction techniques (Figure 1C, D). While nuclei that were isolated with the classical technique showed some fibrous structures around the nuclei, the PNF-extracted nuclei did not. To address the question of how much the perinuclear fraction was contaminated with cytosolic or nuclear content, the marker proteins of the fractions were analyzed by immunoblotting. In order to distinguish the perinuclear fraction from other cellular compartments, cells were lysed, and the nuclei were isolated as described above, after which the PNF was extracted and analyzed (Figures 2,3,4,5). The PNF and other cellular fractions from two cancer cell lines and two mouse embryonic fibroblasts (immortalized and primary fibroblasts) were tested (Table 2).


Dissecting the cell to nucleus, perinucleus and cytosol.

Shaiken TE, Opekun AR - Sci Rep (2014)

Patterns of protein distribution in MDA-MB-435 cells.Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for CDS method; Nuc. Cont is a control for nuclear proteins obtained with classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from cytoplasm. They are not detected in perinuclear and nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques (B) Proteins detected in the cytosol and the perinuclear fraction: proteins were detected as cytosolic proteins with both cellular lysis technique; in addition, these proteins also appeared in the perinuclear fraction by extraction with Buffer B. (C) Proteins of perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. (D) Proteins detected in nuclear and perinuclear fractions: transcription factors (that supposedly belong to the nuclear fraction) also appeared in the perinuclear fraction. (E) Nuclear proteins: proteins were detected in the nuclear fraction. Nuclear proteins were obtained with both new and classical nuclei isolation techniques. The PVDF membranes were cropped into two halves and the high and low molecular weight proteins were shown correspondingly.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017230&req=5

f2: Patterns of protein distribution in MDA-MB-435 cells.Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for CDS method; Nuc. Cont is a control for nuclear proteins obtained with classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from cytoplasm. They are not detected in perinuclear and nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques (B) Proteins detected in the cytosol and the perinuclear fraction: proteins were detected as cytosolic proteins with both cellular lysis technique; in addition, these proteins also appeared in the perinuclear fraction by extraction with Buffer B. (C) Proteins of perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. (D) Proteins detected in nuclear and perinuclear fractions: transcription factors (that supposedly belong to the nuclear fraction) also appeared in the perinuclear fraction. (E) Nuclear proteins: proteins were detected in the nuclear fraction. Nuclear proteins were obtained with both new and classical nuclei isolation techniques. The PVDF membranes were cropped into two halves and the high and low molecular weight proteins were shown correspondingly.
Mentions: A slight difference was visible under the phase contrast microscope between nuclei fractionated with the different nuclear-extraction techniques (Figure 1C, D). While nuclei that were isolated with the classical technique showed some fibrous structures around the nuclei, the PNF-extracted nuclei did not. To address the question of how much the perinuclear fraction was contaminated with cytosolic or nuclear content, the marker proteins of the fractions were analyzed by immunoblotting. In order to distinguish the perinuclear fraction from other cellular compartments, cells were lysed, and the nuclei were isolated as described above, after which the PNF was extracted and analyzed (Figures 2,3,4,5). The PNF and other cellular fractions from two cancer cell lines and two mouse embryonic fibroblasts (immortalized and primary fibroblasts) were tested (Table 2).

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