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Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes.

Prudovsky I, Vary CP, Markaki Y, Olins AL, Olins DE - Nucleus (2012)

Bottom Line: Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation.Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones.This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Maine Medical Center Research Institute, Scarborough, ME, USA.

ABSTRACT
Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

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Figure 1. Immunostaining of interphase and mitotic cells with anti-phosphatidylserine (1H6) compared with epichromatin (PL2–6). (A–F) Deconvolution imaging of PFA-fixed U2OS cells with 1H6 (red) and DAPI (blue). (G–I) Confocal images of colocalized 1H6 (red) and PL2–6 (green) on methanol-fixed NIH 3T3 cells; blue represents TOPRO-3 stained DNA. Images are merged in (I). (J–L) 3-D SIM images of PFA-fixed U2OS cells reacted with 1H6 (J), PL2–6 (K) and PL2–7 (L); antibodies staining (red), DAPI (blue). Higher magnification inserts are shown at the right of each frame, presented top-to-bottom as antibody, DAPI and merge. Scale bars: (A–F), 10 µm; (G–I), 10 µm; (J–L), 5 µm; (J–L inserts), 500 nm.
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Figure 1: Figure 1. Immunostaining of interphase and mitotic cells with anti-phosphatidylserine (1H6) compared with epichromatin (PL2–6). (A–F) Deconvolution imaging of PFA-fixed U2OS cells with 1H6 (red) and DAPI (blue). (G–I) Confocal images of colocalized 1H6 (red) and PL2–6 (green) on methanol-fixed NIH 3T3 cells; blue represents TOPRO-3 stained DNA. Images are merged in (I). (J–L) 3-D SIM images of PFA-fixed U2OS cells reacted with 1H6 (J), PL2–6 (K) and PL2–7 (L); antibodies staining (red), DAPI (blue). Higher magnification inserts are shown at the right of each frame, presented top-to-bottom as antibody, DAPI and merge. Scale bars: (A–F), 10 µm; (G–I), 10 µm; (J–L), 5 µm; (J–L inserts), 500 nm.

Mentions: In order to examine the distribution of chromatin-associated phosphatidylserine during the division cycle, we performed an immunostaining comparison of anti-PS antibody (1H6) with anti-epichromatin antibody (PL2-6) on rapidly growing tissue culture cells (i.e., human U2OS and mouse NIH 3T3). With the use of 1H6 on formaldehyde-fixed (PFA) and Triton X-100 permeabilized U2OS cells, the deconvolution microscopy images (Fig. 1A–F) bore a remarkable resemblance to U2OS cells immunostained with anti-epichromatin PL2-6 (ref. 2, Fig. 2A). For both antibodies, the interphase nuclei showed strong staining adjacent to the INM. During prophase, 1H6 staining was apparent on the most peripheral chromosomal regions, while at metaphase it surrounded the congressed chromosomes. During early anaphase, 1H6 reactivity remained confined to the periphery of chromosomes, whereas by late anaphase it was detectable on trailing chromosome arms. During telophase and G1 phase, the decondensing chromosomes showed only peripheral surface staining, similar to results for interphase epichromatin. We have obtained identical images using 1H6 on U2OS cells fixed with methanol (-20°C, 10 min), indicating that the fixation reagent is not a factor. In addition, we have recently identified a second independently derived purified mouse monoclonal anti-phosphatidylserine (Abcam clone 4B6), which yields identical immunofluorescent staining, compared with 1H6, with either PFA or ethanol fixation (Fig. S1). A more direct demonstration of colocalization of PS within the epichromatin region is presented in Figure 1G–I. Interphase and mitotic chromosomes of methanol-fixed NIH 3T3 cells were reacted first with PL2-6, washed with PBS and reacted with FITC-anti-mouse IgG. Subsequent PBS washes were followed by incubation with an excess of normal mouse IgG, in order to saturate all binding sites on the anti-mouse IgG. Lastly, the slides were incubated with TRITC-1H6, washed, incubated with TOPRO-3 and visualized by confocal microscopy. Supporting images document that the peripheral staining by 1H6 is not a consequence of an antibody-established barrier against diffusion (Fig. S2A). For immunostaining, we normally employ 1H6 at 1:200 dilution; identical, but weaker, images are obtained up to a 1:5400 dilution, making it unlikely that a concentration-dependent “layer” of bound antibody impedes diffusion of 1H6 deeper into the nucleus. A related control experiment (Fig. S2B) demonstrates that there is neither a 1H6 barrier blocking the nuclear penetration of an unrelated mouse mAb (BM28), nor does the strong peripheral FITC staining cause any obvious attenuation of the exciting and emitted light.


Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes.

Prudovsky I, Vary CP, Markaki Y, Olins AL, Olins DE - Nucleus (2012)

Figure 1. Immunostaining of interphase and mitotic cells with anti-phosphatidylserine (1H6) compared with epichromatin (PL2–6). (A–F) Deconvolution imaging of PFA-fixed U2OS cells with 1H6 (red) and DAPI (blue). (G–I) Confocal images of colocalized 1H6 (red) and PL2–6 (green) on methanol-fixed NIH 3T3 cells; blue represents TOPRO-3 stained DNA. Images are merged in (I). (J–L) 3-D SIM images of PFA-fixed U2OS cells reacted with 1H6 (J), PL2–6 (K) and PL2–7 (L); antibodies staining (red), DAPI (blue). Higher magnification inserts are shown at the right of each frame, presented top-to-bottom as antibody, DAPI and merge. Scale bars: (A–F), 10 µm; (G–I), 10 µm; (J–L), 5 µm; (J–L inserts), 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Figure 1. Immunostaining of interphase and mitotic cells with anti-phosphatidylserine (1H6) compared with epichromatin (PL2–6). (A–F) Deconvolution imaging of PFA-fixed U2OS cells with 1H6 (red) and DAPI (blue). (G–I) Confocal images of colocalized 1H6 (red) and PL2–6 (green) on methanol-fixed NIH 3T3 cells; blue represents TOPRO-3 stained DNA. Images are merged in (I). (J–L) 3-D SIM images of PFA-fixed U2OS cells reacted with 1H6 (J), PL2–6 (K) and PL2–7 (L); antibodies staining (red), DAPI (blue). Higher magnification inserts are shown at the right of each frame, presented top-to-bottom as antibody, DAPI and merge. Scale bars: (A–F), 10 µm; (G–I), 10 µm; (J–L), 5 µm; (J–L inserts), 500 nm.
Mentions: In order to examine the distribution of chromatin-associated phosphatidylserine during the division cycle, we performed an immunostaining comparison of anti-PS antibody (1H6) with anti-epichromatin antibody (PL2-6) on rapidly growing tissue culture cells (i.e., human U2OS and mouse NIH 3T3). With the use of 1H6 on formaldehyde-fixed (PFA) and Triton X-100 permeabilized U2OS cells, the deconvolution microscopy images (Fig. 1A–F) bore a remarkable resemblance to U2OS cells immunostained with anti-epichromatin PL2-6 (ref. 2, Fig. 2A). For both antibodies, the interphase nuclei showed strong staining adjacent to the INM. During prophase, 1H6 staining was apparent on the most peripheral chromosomal regions, while at metaphase it surrounded the congressed chromosomes. During early anaphase, 1H6 reactivity remained confined to the periphery of chromosomes, whereas by late anaphase it was detectable on trailing chromosome arms. During telophase and G1 phase, the decondensing chromosomes showed only peripheral surface staining, similar to results for interphase epichromatin. We have obtained identical images using 1H6 on U2OS cells fixed with methanol (-20°C, 10 min), indicating that the fixation reagent is not a factor. In addition, we have recently identified a second independently derived purified mouse monoclonal anti-phosphatidylserine (Abcam clone 4B6), which yields identical immunofluorescent staining, compared with 1H6, with either PFA or ethanol fixation (Fig. S1). A more direct demonstration of colocalization of PS within the epichromatin region is presented in Figure 1G–I. Interphase and mitotic chromosomes of methanol-fixed NIH 3T3 cells were reacted first with PL2-6, washed with PBS and reacted with FITC-anti-mouse IgG. Subsequent PBS washes were followed by incubation with an excess of normal mouse IgG, in order to saturate all binding sites on the anti-mouse IgG. Lastly, the slides were incubated with TRITC-1H6, washed, incubated with TOPRO-3 and visualized by confocal microscopy. Supporting images document that the peripheral staining by 1H6 is not a consequence of an antibody-established barrier against diffusion (Fig. S2A). For immunostaining, we normally employ 1H6 at 1:200 dilution; identical, but weaker, images are obtained up to a 1:5400 dilution, making it unlikely that a concentration-dependent “layer” of bound antibody impedes diffusion of 1H6 deeper into the nucleus. A related control experiment (Fig. S2B) demonstrates that there is neither a 1H6 barrier blocking the nuclear penetration of an unrelated mouse mAb (BM28), nor does the strong peripheral FITC staining cause any obvious attenuation of the exciting and emitted light.

Bottom Line: Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation.Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones.This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Maine Medical Center Research Institute, Scarborough, ME, USA.

ABSTRACT
Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

Show MeSH