Three-Dimensional Cellular Raman Analysis: Evidence of Highly Ordered Lipids Within Cell Nuclei

Abstract

Striking levels of spatial organization exist among and within interphase cell chromosomes, raising the possibility that other nuclear molecular components may also be organized in ways that facilitate nuclear function. To further examine molecular distributions and organization within cell nuclei, we utilized Raman spectroscopy to map distributions of molecular components, with a focus on cellular lipids. Although the vast majority of cellular lipids are associated with membranes, mapping the 2870/2850 cm^-1 lipid peak ratios revealed that the most highly ordered lipids within interphase cells are found within cell nuclei. This finding was seen in cells from multiple tissue types, noncancerous cells, and in cancer cell lines of different metastatic potential. These highly ordered lipids colocalize with nuclear chromatin, are present throughout the nuclear volume, and remain colocalized with chromatin through mitosis, when the nuclear envelope has dissociated. Phosphatidylinositol is a major component of the highly ordered lipids. The presence of phosphatidylinositol and other lipids in the nuclear interior is well established, but their highly ordered packing has not been reported and represents a unique finding. The molecular interactions involved in the formation and maintenance of these highly ordered lipids, and their potential effects on nuclear activities, remain to be discovered.

Keywords: DNA; Raman spectral analysis; cell nucleus; chromatin; nuclear lipids; phospholipids.

Figure 1.

Raman spectra at various points within a human osteosarcoma MG-63 cell. The crosshairs on the cell images (A, C, E, G) represent the points within the cell where the Raman spectra (B, D, F, H) were taken. A and B, plasma membrane. C and D, cell cytoplasm. E and F, nuclear membrane. G and H, within the nucleus at the edge of nucleolus.

Figure 2.

The distribution of highly ordered lipids within interphase osteosarcoma cells. Laser Raman spectra were collected on MG-63 human osteosarcoma cells and the distributions of lipid methyl groups in different conformations was mapped. Panel A represents a white light image. Panel B represents the distribution of 2850 cm⁻¹ Raman peak amplitudes, which represents lipid methyl groups in the gauche conformation that occur in more loosely packed lipids. Panel C represents the distribution of 2870 cm⁻¹ Raman peak amplitudes, which represent lipid methyl groups in the trans conformation, which allows more tightly packed, ordered lipids. Panel D represents the distribution of the 2870/2850 cm⁻¹; the highest values of this ratio represent the regions of highest lipid packing order. The most highly ordered lipids are present throughout the nuclear interior and appear associated with nuclear chromatin.

Figure 3.

The regions within nuclei containing the highest concentrations of highly ordered lipids are the nucleoli. MG-63 cells were analyzed by laser Raman spectroscopy to identify regions of the most highly ordered lipids, and were also stained with Nuclear Red and Nucleolar Green stains to identify nuclear and nucleolar regions, respectively. Panel A represents a white light image, and panel B represents the distribution of the ordered lipid ratio (2870/2850 cm⁻¹) in the cell shown in panel A. Panel C represents a white light image of MG-63 cells, and panel D represents the cells in panel C stained with nuclear red. Panel E represents a white light image of MG-63 cells, and panel F represents the cells shown in panel E stained with nucleolar green.

Figure 4.

The distribution of highly ordered lipids within breast epithelial and breast cancer cells. Laser Raman analysis was performed on noncancerous MCF-10A (Fig. 3A) human breast epithelial cells, noninvasive MCF-7 (Fig. 3B) human breast adenocarcinoma cells, and highly invasive MDA-MB231 (Fig 3C) human breast adenocarcinoma cells. For each cell line, panel a represents a white light image, panel b represents the distribution of the 2850 cm⁻¹ lipid methyl peak (blue), panel c represents the distribution of the 2870 cm⁻¹ lipid methyl peak (green), and panel d represents the distribution of the 2870/2850 cm⁻¹ (red, highly ordered lipids).

Figure 5.

The distribution of cellular protein as mapped by the 1650 cm⁻¹ amide shift. Because the entire Stokes shifted Raman spectra (from 300 to 3100 cm⁻¹) could not be collected using a single diffraction grating setting, data were collected twice within a single defined volume using two settings, one collecting the lower end of the spectrum (300 to 2100 cm⁻¹), the other collecting the high end of the spectrum (1600 to 3100 cm⁻¹). This approach produced two sets of spectra covering a defined volume which overlapped in the 1600 to 1700 cm⁻¹ region. The amide band at 1650 cm⁻¹ appeared in both spectra and was used to compare or normalize the lower and higher region spectra as needed. Panel A represents a white light image of an MG-63 cell, panel B represents the distribution of the 1650 cm⁻¹ peak using data from the lower region spectrum and panel C represents the distribution of the 1650 cm⁻¹ peak using data from the higher region spectrum. Representative Raman spectra covering the lower and upper ends are shown, with the 1650 cm⁻¹ peak in each spectrum circled.

Figure 6.

The distribution of highly ordered lipids during mitosis. MCF-7 cells were synchronized in early mitosis by overnight incubation with nocodazole, released to allow mitotic entry, and then fixed for laser Raman spectra collection. In the MCF-7 cell undergoing cytokinesis, panel A represents the white light image, panel B represents the distribution of the 2850 cm⁻¹ lipid methyl peak (green), panel C represents the distribution of the 2870 cm⁻¹ lipid methyl peak (blue), panel D represents the distribution of the 2870/2850 cm⁻¹ (red), and panel E represents the 2870/2850 cm⁻¹ (red) juxtaposed over the 2850 cm⁻¹ lipid methyl peak (green).

Figure 7.

The distribution of phosphatidylinositol and the distribution of the highly ordered lipids in interphase MG-63 and MCF-7 cells. In the first group of images showing an MG-63 cell (7A), panel a represents the white light image, panel b represents the 1093 cm⁻¹ DNA peak (red), and panel c represents the 779 cm⁻¹ phosphatidylinositol peak (blue). In the second group of images showing an MCF-7 cell (7B), panel a represents the white light image, panel b represents the 1093 cm⁻¹ DNA peak (red), and panel c represents the 779 cm⁻¹ phosphatidylinositol peak (blue). In the third group of images showing an MG-63 cell (7C), panel a represents the white light image, panel b represents the 779 cm⁻¹ phosphatidylinositol peak (blue), panel C represents the 2870/2850 cm⁻¹ ratio of trans to gauche lipid methyl peaks (red).

Figure 8.

The distribution of phosphatidylinositol and highly ordered lipids in MCF-7 cells during mitosis. In the first group of images representing MCF-7 cells treated with nocodazole (8A), panel a represents a white light image, panel b represents the 1093 cm⁻¹ DNA peak (red), panel c represents the 779 cm⁻¹ phosphatidylinositol peak (blue), panel d represents the 2850 cm⁻¹ lipid methyl gauche conformation peak (blue), panel e represents the 2870 cm⁻¹ lipid methyl trans conformation peak (green), and panel f represents the 2870/2850 cm⁻¹ ratio of trans to gauche lipid methyl peaks (red). In the second group of images representing MCF-7 cells also treated with nocodazole (8B), panel a represents a white light image, panel b represents the 1093 cm⁻¹ DNA peak (red), panel c represents the 779 cm⁻¹ phosphatidylinositol peak (blue), panel d represents the 2850 cm⁻¹ lipid methyl gauche conformation peak (blue), panel e represents the 2870 cm⁻¹ lipid methyl trans conformation peak (green), and panel f represents the 2870/2850 cm⁻¹ ratio of trans to gauche lipid methyl group peaks (red). In the third group of images representing MCF-7 cells selected during mitosis using the mitotic shake-off technique (8C), panel a represents a white light image, panel b represents the 1093 cm⁻¹ DNA peak (red), panel c represents the 779 cm⁻¹ phosphatidylinositol peak (blue), panel d represents the 2850 cm⁻¹ lipid methyl gauche conformation peak (green), panel e represents the 2870 cm⁻¹ lipid methyl trans conformation peak (blue), and panel f represents the 2870/2850 cm⁻¹ ratio of trans to gauche lipid methyl group peaks (red).