Stem cells. Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness

Abstract

By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.

Fig. 1. Asymmetric apportioning of aged mitochondria during cell division

(A) Schematic of the labeling strategy using paGFP. (B) Analysis of apportioning of a fluorescent lipid dye, and of green fluorescence marking five paGFP-targeted organelles during cell division in epithelial (morphologically flat, arrowhead), and stem-like (morphologically round, arrow) cells in cultures of human mammary epithelium cells (hMECs). P1 and P2 indicate daughter cells, with P1 being the daughter receiving more of the targeted organelle. Inheritance indicates fluorescence of daughter cells relative to that of the mother cell scaled to 1. (C) Dynamics of mitochondrial apportioning in round SLCs and flat epithelial cells. Representative divisions are shown with mitochondria in green (paGFP-Omp25) and the lipid dye (PKH26) in red. Images are frame captures from one hour before and after division, and fluorescence intensity per cell is plotted at one-hour intervals. (D) Analysis of asymmetric apportioning as a function of label age. SLCs dividing more than 10 hours after label activation show increasing asymmetric apportioning of mitochondria (Omp25), but not a chromatin label (H2B). Each data point represents an individual cell division. (**p<0.01, ***p<0.001, t-test)

Fig. 2. Age-dependent segregation and subcellular localization of mitochondria

(A) Schematic of the labeling strategy using Snap-tag chemistry. (B–C) Analysis of mitochondrial outer membrane (B) and inner membrane (C) inheritance upon cell division. Red and green sections of bars represent the old and young labels, respectively. Values were scaled so that total intensity (red+green) of the mother cell is 1 (n=5). Representative division occurring at 6 hours after the second (green) label is shown. Percent values represent the average of five divisions. Original magnification 40×. (D) Confocal microscopy of a cell with ≥50 hour-old and 0 to 1 hour-old mitochondrial Snap-Omp25 labeled red and green, respectively. Mitochondrial network contains domains with different levels of enrichment for the old proteins. Mitochondrial domains enriched with old proteins (arrow heads) are not associated with autophagosomes detected by immunofluorescence for LC3B (purple) (63×, 2 μm Z-section). (E) Localization of old (red) and young (green) mitochondria (Snap-Omp25) 10 hours after labeling in an undivided cell. Squares mark regions used for measurements of the perinuclear and peripheral intensities in frames captured 10 hours after labeling for n=3 (cells imaged from three separate labeling experiments) (*p<0.05, **p<0.01, t-test).

Fig. 3. Stemness properties of daughter cells receiving younger mitochondria

(A) FACS-mediated isolation of cell populations with high and low contents of old mitochondria (Pop1 and Pop2, respectively). Images show representative populations after 1 and 3 days in culture. (B) Mammosphere forming capacity of Pop1 and Pop2 cells for n=5 (scale bar 50μm, **p<0.01, t-test).

Fig. 4. Effects of mitochondrial quality control on asymmetric apportioning of old mitochondria during cell division

(A) FACS analyses of mitochondrial apportioning in cells with defective mitochondrial quality control induced by siRNA-mediated depletion of Parkin (siParkin) or pharmacological inhibition of mitochondrial fission (mDivi-1). Table presents the percentages of cells in the two populations for n=3. (B) Mammosphere-forming capacity of cells in Pop1 and Pop2 are equal following siRNA Parkin and mDivi-1 (n=3). (C) Localization of old mitochondria following treatment with mDivi-1. Images are frame captures at start (0 hours) and 5 hours after mDivi-1 administration. Original magnification 63×. (*p<0.05, **p<0.01, t-test).