Subcellular redistribution and mitotic inheritance of transition metals in proliferating mouse fibroblast cells

Abstract

Synchrotron X-ray fluorescence microscopy of non-synchronized NIH 3T3 fibroblasts revealed an intriguing redistribution dynamics that defines the inheritance of trace metals during mitosis. At metaphase, the highest density areas of Zn and Cu are localized in two distinct regions adjacent to the metaphase plate. As the sister chromatids are pulled towards the spindle poles during anaphase, Zn and Cu gradually move to the center and partition into the daughter cells to yield a pair of twin pools during cytokinesis. Colocalization analyses demonstrated high spatial correlations between Zn, Cu, and S throughout all mitotic stages, while Fe showed consistently different topographies characterized by high-density spots distributed across the entire cell. Whereas the total amount of Cu remained similar compared to interphase cells, mitotic Zn levels increased almost 3-fold, suggesting a prominent physiological role that lies beyond the requirement of Zn as a cofactor in metalloproteins or messenger in signaling pathways.

Fig. 1

Intracellular elemental redistribution in non-synchronized NIH 3T3 cells during mitosis. Top row: Fluorescence micrographs of cells stained with Hoechst 33258, a DNA selective fluorescent probe that highlights the chromosome morphology for assigning individual mitotic stages. 2nd-6th rows: Subcellular distribution of phosphorus (P), sulfur (S), iron (Fe), copper (Cu), and zinc (Zn) for each cell (top row) visualized by SXRF raster scans with excitation at 10 keV and 0.3 µm step size. All false-color maps were normalized to the maximum elemental density indicated at the top left corner (units of 10 ng/cm²). All scale bars correspond to 10 µm.

Fig. 2

Subcellular distribution of areas with the highest densities of Zn and Cu during mitosis. The integrated elemental content of the areas highlighted in red corresponds to 30% of the total cellular content of Zn (top row) or Cu (bottom row). The depicted cells are identical with those in Fig. 1 at the respective mitotic stages.

Fig. 3

Colocalization analyses of the subcellular distribution of Zn in relation to S, P, and Cu for metaphase (panel A) and anaphase (panel B) cells. Left column: False-color overlay of the SXRF density maps of Zn (green) and selected elements (red) as indicated in each panel. Colocalized areas appear in yellow. Scale bars: 10 µm. Right column: Correlation analyses based on scatter plots of the respective elemental densities at each pixel within the cellular area. The resulting Pearson correlation coefficients are displayed in the top left corner of each scatter plot. Linearly correlated pixels (sky blue) were identified in the scatter plot and the corresponding subcellular locations highlighted in the gray-scale elemental map. In select cases, a non-correlated subset was also plotted (orange pixels).

Fig. 4

Intracellular elemental distributions in interphase NIH 3T3 cells. Top row: confocal fluorescence micrographs of cells labeled with the cell cycle indicator FUCCI for assigning individual interphase stages (red: G1 phase; mixed red/green: G1/S phase; green: G2). 2nd–6th rows: Subcellular distribution of phosphorus (P), sulfur (S), iron (Fe), copper (Cu), and zinc (Zn) for each top row cell visualized by SXRF raster scans with excitation at 10 keV and 0.3 µm step size. All false-color maps were normalized to the maximum elemental density. Scale bars: 20 µm.

Fig. 5

Comparison of the x-ray emission spectra for cells occurring at selected stages of the cell cycle. Pixel-by-pixel emission spectra were integrated over the entire cell area and normalized to the intensity of the sulfur Kα emission at 2.31 keV. Each spectrum represents the averaged spectra of three independent raster scans of different cells occurring at the same stage of the cell cycle.

Fig. 6

Colocalization analyses of the subcellular distribution of Zn in relation to S, P, and Cu for G1 (panel A) and G2 (panel B) interphase cells. Left column: False color overlay of the SXRF density maps of Zn (green) and selected elements (red) as indicated in each panel. Colocalized areas appear in yellow. Scale bars: 20 µm. Right column: Correlation analyses based on scatter plots of the respective elemental densities at each pixel within the cellular area. The resulting Pearson correlation coefficients are displayed in the top left corner of each scatter plot. Linearly correlated pixels (sky blue) were identified in the scatter plot and the corresponding subcellular locations highlighted in the gray-scale elemental map. In select cases, a non-correlated subset was also plotted (orange pixels).

Fig. 7

Intensity correlation analysis (ICA) for the subcellular distribution of Zn in relation to S and P at various stages of the cell cycle. The SXRF data sets for select cells occurring in G1 (left), metaphase (middle), and anaphase (right) were subjected to ICA to evaluate the Zn-S (panel A) and Zn-P (panel B) spatial relationships. Scatter plots are shown for the pixel-by-pixel correlation of the Zn densities (Zn_i) with the product (Zn_i-Zn)(S_i-S), where Si corresponds to the S density at pixel i, and Zn and S represent the average elemental densities within the cellular boundaries. Area densities with above average elemental content are color-coded in blue for synchronously varying pixels and orange for segregated pixels. Below average segregated pixels are plotted in magenta.

Fig. 8

Comparison of the Pearson correlation coefficients for the colocalization of selected elements at individual stages of the cell cycle. The corresponding Pearson coefficients were converted into a color-coded heat map using a non-linear scale shown on the right, which highlights the most significant correlations (>0.9) in hues of blue-green.