Figure 4.
Asymmetric TMRM inheritance predicts mitochondrial activation. (A) Experimental design. (B) Dividing CD49fHSC with quantification of TMRM inheritance and dynamics. Representative frames of CD49fHSCs with a-/symmetric TMRM inheritance (trees 4 and 2 in panel C, respectively). Cells with symmetric inheritance of TMRM produce comparable levels TMRM in both daughter cells. With asymmetric inheritance, daughters receiving more TMRM upregulate mitochondrial activity (blue arrowhead). n = 4 independent experiments. Scale bar: 10 µm. (C) Representative quantification of TMRM inheritance and dynamics in paired daughter cells. Daughters receiving more TMRM have more future mitochondrial activity. Fold sister differences early after division are indicated, respectively. (D,E) Heatmap and clustering (top) and cluster frequency (bottom) of paired daughter cell TMRM dynamics (D) or CD71 production (E) after a-/symmetric inheritance of TMRM. Each row represents a CD49fHSC daughter pair (#1 and #2). Daughter #1 receives more TMRM during asymmetric inheritance and later has more active mitochondria (cluster 3 with mean paired daughter difference: −645 ± 112; clusters 1/2 with 847 ± 39/80 ± 208). TMRM inheritance does not predict symmetric (cluster 1; mean paired daughter difference: 57 ± 66) or asymmetric (clusters 2 and 3; −572 ± 33 and 382 ± 74) CD71 production. Lower left, mean TMRM and CD71 fluorescence intensities of a-/symmetric paired daughter cell clusters, respectively, mean ± SD. n = 4 independent experiments, 110 and 37 analyzed symmetric (<1.1-fold) and asymmetric (>1.5-fold) divisions, respectively. (F-H) Quantification of CD71 production (F) mitochondrial activity (G) and CD49fHSC daughter cell lifetimes (H) after a-/symmetric TMRM inheritance. TMRM inheritance predicts mitochondrial activity in CD49fHSCs daughter cells, but not CD71 production and daughter cell lifetimes. Box-plot elements: center line, median; box limits, upper and lower quartiles; Tukey’s 1.5× interquartile range; points, outlier. n = 4 independent experiments, 110 and 37 analyzed symmetric (<1.1-fold) and asymmetric (>1.5-fold) divisions, respectively. (I) Overall frequency of a-/symmetric LysoBrite/TMRM co-/anti-inheritance and future paired daughter cell mitochondrial activity (TMRM area under the curve). Antisegregation of TMRM and lysosomes correlates with asymmetric mitochondria activity of CD49fHSC daughters. (J) Frequency of a-/symmetric lysosome and TMRM division modes of paired daughter cells with only asymmetric mitochondrial activity. Asymmetric antisegregation of TMRM and lysosomes correlates with asymmetric mitochondrial activity in paired daughter cells. n = 8 independent experiments with 647 CD49fHSC divisions total.

Asymmetric TMRM inheritance predicts mitochondrial activation. (A) Experimental design. (B) Dividing CD49fHSC with quantification of TMRM inheritance and dynamics. Representative frames of CD49fHSCs with a-/symmetric TMRM inheritance (trees 4 and 2 in panel C, respectively). Cells with symmetric inheritance of TMRM produce comparable levels TMRM in both daughter cells. With asymmetric inheritance, daughters receiving more TMRM upregulate mitochondrial activity (blue arrowhead). n = 4 independent experiments. Scale bar: 10 µm. (C) Representative quantification of TMRM inheritance and dynamics in paired daughter cells. Daughters receiving more TMRM have more future mitochondrial activity. Fold sister differences early after division are indicated, respectively. (D,E) Heatmap and clustering (top) and cluster frequency (bottom) of paired daughter cell TMRM dynamics (D) or CD71 production (E) after a-/symmetric inheritance of TMRM. Each row represents a CD49fHSC daughter pair (#1 and #2). Daughter #1 receives more TMRM during asymmetric inheritance and later has more active mitochondria (cluster 3 with mean paired daughter difference: −645 ± 112; clusters 1/2 with 847 ± 39/80 ± 208). TMRM inheritance does not predict symmetric (cluster 1; mean paired daughter difference: 57 ± 66) or asymmetric (clusters 2 and 3; −572 ± 33 and 382 ± 74) CD71 production. Lower left, mean TMRM and CD71 fluorescence intensities of a-/symmetric paired daughter cell clusters, respectively, mean ± SD. n = 4 independent experiments, 110 and 37 analyzed symmetric (<1.1-fold) and asymmetric (>1.5-fold) divisions, respectively. (F-H) Quantification of CD71 production (F) mitochondrial activity (G) and CD49fHSC daughter cell lifetimes (H) after a-/symmetric TMRM inheritance. TMRM inheritance predicts mitochondrial activity in CD49fHSCs daughter cells, but not CD71 production and daughter cell lifetimes. Box-plot elements: center line, median; box limits, upper and lower quartiles; Tukey’s 1.5× interquartile range; points, outlier. n = 4 independent experiments, 110 and 37 analyzed symmetric (<1.1-fold) and asymmetric (>1.5-fold) divisions, respectively. (I) Overall frequency of a-/symmetric LysoBrite/TMRM co-/anti-inheritance and future paired daughter cell mitochondrial activity (TMRM area under the curve). Antisegregation of TMRM and lysosomes correlates with asymmetric mitochondria activity of CD49fHSC daughters. (J) Frequency of a-/symmetric lysosome and TMRM division modes of paired daughter cells with only asymmetric mitochondrial activity. Asymmetric antisegregation of TMRM and lysosomes correlates with asymmetric mitochondrial activity in paired daughter cells. n = 8 independent experiments with 647 CD49fHSC divisions total.

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