Mitochondrial localization and nuclear polarization are synchronized during terminal maturation of mouse erythroblasts. (A) Snapshots of live confocal imaging of purified bone marrow erythroblasts to identify mitochondrial localization during increasing stages of maturation. Cells were stained with DRAQ5 (nucleus, blue), MitoTracker Green (MTG; mitochondria, green), and TER119 (erythroid marker, red). Arrows show mitochondrial localization. (B) Plots of Azimuthal average (also supplemental Figure 1C) integrate the signal intensity of TOM20 (mitochondria, red) around the center of the cell, displaying the normalized intensity around the whole cell counterclockwise starting at 0 degrees (evenly distributed [top], localized mitochondria [bottom]). (C-D) Representative confocal imaging of mitochondrial localization during erythroblast maturation was captured on Leica DM550 and analyzed by ImageJ; TOM20 (mitochondria, red) and DAPI (nucleus, blue) in erythroblasts (bar, 5 μm) (C), BrightField (BF; gray); and quantification of mitochondrial localization index to a specific area of the cell (D). The LI is calculated (data supplement; supplemental Figure 1C) such that mitochondria localized in 1 area of the cell that display fewer peaks including all of the fluorescence result in greater LI, with gate 3 (supplemental Figure 1B) showing the highest index of mitochondrial localization (supplemental Figure 1). The peaks of intensity were defined as: height, minimal 50% of the distance from the minimum to the maximum intensity above baseline; width, >5 degrees. (E) ImageStream analyses of gate 2 and 3 cells (supplemental Figure 2D) stained with TER119 (erythroid marker, yellow) and MTG (mitochondria, green) are shown with either evenly distributed (top) or localized mitochondria (bottom). The distribution of mitochondria was determined quantitatively using morphology-masked mitochondria based on circularity, measured as the average radius divided by the variance in radius, and the Δ_mitoC, measured as the distance from the center of the mitochondrial networks from the center of the cell. (F) The scatter plot of erythroblasts (from panel E) with either localized mitochondria (low circularity_mito, high Δ_mitoC) or evenly distributed mitochondria (high circularity_mito, low Δ_mitoC). Plotted ratio of localized to evenly distributed mitochondria compare mitochondrial localization in progressively maturing erythroblast stages (right panel). (G) Representative cells from ImageStream analysis identifying gate 3 erythroblasts stained with MTG (mitochondria, green), TER119 (erythroid marker, yellow), and DRAQ5 (nucleus, red), as enucleating (high Δ_nucC) or nonenucleating (low Δ_nucC) based on the distance between the center of the nucleus and the cell. (H) The percentage of enucleating cells in erythroblasts with either localized or evenly distributed mitochondria was determined. Enucleating cells were gated on cells with high Δ Centroid between the nucleus and the maturing erythroblast. (I) The reciprocal analysis was performed by determining the frequency of erythroblasts with localized mitochondria in enucleating (E) G3 erythroblasts or nonenucleating (NE) G3 erythroblasts. Mean ± standard deviation (n ≥ 3). *P < .05, **P < .01 by Student t test (C-D,H-I) or 1-way analysis of variance followed by Tukey multiple comparisons test (F).