Dissecting the cell to nucleus, perinucleus and cytosol - PubMed (original) (raw)

Dissecting the cell to nucleus, perinucleus and cytosol

Tattym E Shaiken et al. Sci Rep. 2014.

Erratum in

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.

PubMed Disclaimer

Figures

Figure 1

Figure 1. 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.

Figure 2

Figure 2. 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.

Figure 3

Figure 3. Patterns of protein distribution in HeLa cells.

Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for the CDS method; Nuc. Cont is a control for nuclear proteins obtained with the classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for the CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from the cytoplasm. They are not detected in the perinuclear and the 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 perinuclear fraction by buffer B extraction. p53 protein was detected with long exposure. (D) Proteins detected in the nuclear and the perinuclear fractions: transcription factor CREB appeared in both fractions. (E) Nuclear proteins: proteins were detected in nuclear fraction. Nuclear proteins were obtained with 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.

Figure 4

Figure 4. Patterns of protein distribution in MEF cells.

Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for the 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 the CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from the 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 the perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. CBP protein was detected with long exposure. (D) Proteins detected in nuclear and perinuclear fractions: transcription factor CREB appeared in both fractions, in addition Ras and p53 proteins were detected. (E) Nuclear proteins: Nuclear proteins were obtained with 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.

Figure 5

Figure 5. Patterns of protein distribution in Primary MEF cells.

Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for the CDS method; Nuc. Cont is a control for nuclear proteins obtained with the classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for the CDS method. (A) Proteins of cytosol: proteins extracted by regular lysis buffer and Buffer A from the cytoplasm. They are not detected in the perinuclear and the nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques. (B) Proteins detected in the cytosol and the perinuclear fractions: 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 the perinuclear fraction: proteins are detected only in the perinuclear fraction by buffer B extraction. Ras, p53 and CBP proteins were detected with long exposure. (D) Proteins detected in the nuclear and the perinuclear fractions: transcription factor CREB appeared in both the nuclear and the the perinuclear fractions. (E) Nuclear proteins: proteins were detected in nuclear fraction. Nuclear proteins were obtained with 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.

Figure 6

Figure 6. Model view of the perinucleus of cell.

The nuclear boundary of the perinucleus is limited with the nuclear lamina where the N termini of SUN1 and SUN2 form a network with the nuclear lamina. The C termini of SUN proteins interact with the KASH domains of nesprins, that are located at the outer nuclear membrane, where it conntects to plectins (LINC complex). Plectin interacts with intermediate filaments, microtubules and the actin cytoskeleton. This filamentous region on the cytoplasmic side represents the outer boundary of the perinucleus. Note that the Sun 2 proteins form the nuclear boundaries and Plectin 1, from the cytosolic borders, were partially distributed in PNF, representing delimiting boundaries of the perinucleus. The core nucleus embedded into nucleoplasm, and supported by rigid lamin structure, does not collapse after extracting the perinucleus by CDS technique. KEY: CHRM – chromatin, IF – intermediate filaments, INM – inner nuclear membrane, LN – lamin, MT – microtubules, NPC – nuclear pore complex, Nn – Nesprin proteins, ONM – outer nuclear membrane, PL – plectin proteins, PNS – perinuclear space, Sn – Sun proteins.

Similar articles

Cited by

References

    1. Watson M. L. The nuclear envelope; its structure and relation to cytoplasmic membranes. J. Biophys. Biochem. Cytol. 1, 257–270 (1955). - PMC - PubMed
    1. Blose S. H. & Chacko S. Rings of intermediate (100 A) filament bundles in the perinuclear region of vascular endothelial cells. Their mobilization by colcemid and mitosis. J. Cell. Biol. 70, 459–466 (1976). - PMC - PubMed
    1. Bild A. H., Turkson J. & Jove R. Cytoplasmic transport of Stat3 by receptor-mediated endocytosis. EMBO J. 21, 3255–3263 (2002). - PMC - PubMed
    1. Toy-Miou-Leong M., Cortes C. L., Beaudet A., Rostene W. & Forgez P. Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation. J. Biol. Chem. 279, 12636–12646 (2004). - PubMed
    1. Nakamura K., Senda T., Sato K., Mori S. & Moriyama M. Accumulation of BCL10 at the perinuclear region is required for the BCL10-mediated nuclear factor-kappa B activation. Pathobiology. 72, 191–202 (2005). - PubMed

Publication types

MeSH terms

LinkOut - more resources