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. 2018 Mar 13;10(3):684-692.
doi: 10.1016/j.stemcr.2018.01.005. Epub 2018 Feb 8.

A Non-apoptotic Function of MCL-1 in Promoting Pluripotency and Modulating Mitochondrial Dynamics in Stem Cells

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Free PMC article

A Non-apoptotic Function of MCL-1 in Promoting Pluripotency and Modulating Mitochondrial Dynamics in Stem Cells

Megan L Rasmussen et al. Stem Cell Reports. .
Free PMC article

Abstract

Human pluripotent stem cells (hPSCs) maintain a highly fragmented mitochondrial network, but the mechanisms regulating this phenotype remain unknown. Here, we describe a non-cell death function of the anti-apoptotic protein, MCL-1, in regulating mitochondrial dynamics and promoting pluripotency of stem cells. MCL-1 is induced upon reprogramming, and its inhibition or knockdown induces dramatic changes to the mitochondrial network as well as loss of the key pluripotency transcription factors, NANOG and OCT4. Aside from localizing at the outer mitochondrial membrane like other BCL-2 family members, MCL-1 is unique in that it also resides at the mitochondrial matrix in pluripotent stem cells. Mechanistically, we find MCL-1 to interact with DRP-1 and OPA1, two GTPases responsible for remodeling the mitochondrial network. Depletion of MCL-1 compromised the levels and activity of these key regulators of mitochondrial dynamics. Our findings uncover an unexpected, non-apoptotic function for MCL-1 in the maintenance of mitochondrial structure and stemness.

Keywords: DRP-1; MCL-1; OPA1; apoptosis; mitochondria; mitochondrial dynamics; pluripotency; pluripotent stem cells.

Figures

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Figure 1
Figure 1
hESCs Engage Rapid Apoptosis after DNA Damage, which Can Be Reversed upon Cell Differentiation by MCL-1 Downregulation (A) MCL-1 protein expression is significantly decreased in EBs. (B) hESCs were treated with GX15-070 or ABT-737 ± 20 μM etoposide. Cell survival was quantified each hour after etoposide treatment. (C) GX15-070 treatment of hESCs results in decreased expression of NANOG and OCT4. (D) Immunofluorescence images (63×) show decreased staining for OCT4 (green) when treated with GX15-070. Nuclei: Hoechst 33258. Scale bar, 10 μm. (E) In contrast, increasing treatments of ABT-737 do not affect OCT4 or NANOG protein levels. (F) Treatment of hESCs with 500 nM MIM-1 results in lowered expression of NANOG and OCT4. (G and H) Immunofluorescence (20×) shows decreased expression of NANOG and OCT4. Error bars represent ±SD for three independent experiments. Scale bar, 100 μm. See also Figure S1.
Figure 2
Figure 2
MCL-1 Is Highly Expressed in hESCs and Maintains Mitochondrial Fission (A and B) MCL-1 protein expression is increased in hiPSCs (A) and hESCs (B) when compared with human fibroblasts (hFibrob). Bar graphs show quantification of band intensity relative to hFibrob β-actin. (C) hESCs were treated with 500 nM MIM-1, inducing mitochondrial elongation. Cytochrome c staining (Cyt c) depicts mitochondria (63×). Scale bar, 10 μm. (D) Quantification of cells with elongated mitochondria in (C). (E and F) MIM-1 treatment (500 nM) in hESCs results in p-DRP-1 S616 downregulation. Band density was quantified relative to control DMSO. All error bars represent ±SD in at least three independent experiments. See also Figure S2.
Figure 3
Figure 3
MCL-1 Inhibition Results in Elongated Mitochondria and Low Expression of Active DRP-1 (A) Transmission electron microscopy images showing elongated mitochondrial morphology in hESCs after MCL-1 downregulation. Scale bar, 500 nm. (B) Knockdown of MCL-1 results in lowered expression of OCT4 and p-DRP-1 S616. (C) Quantification of western blots (WBs) in (B). Error bars represent ±SD for at least three separate experiments. (D) Representation of murine constructs encoding MCL-1. (E) hESCs were treated with BMP4, then transfected with Mcl-1WT, Mcl-1OM, or Mcl-1Matrix. OCT4 expression was quantitated. Error bars represent ±SD for three independent experiments. See also Figure S3.
Figure 4
Figure 4
MCL-1 Regulates Mitochondrial Dynamics through Interaction with DRP-1 and OPA1 (A) hiPSCs expressing EGFP-MCL-1 or control EGFP and DsRed-mito. Scale bar, 2 μm. (B) Fluorescence intensity plots show co-localization of EGFP-MCL-1 and DsRed-mito. Arrow indicates location of the line used for fluorescence intensity by line scan. (C and D) PLA of cells treated for 6 hr with or without 100 nM S63845 (MCL-1i). Representative Z-stack images are shown. Red indicates MCL-1:OPA1 (C) and MCL-1:DRP-1 (D) proximity; blue indicates Hoechst 33258. Average puncta/cell was quantitated for at least 300 cells/sample (each sample done in triplicate). Error bars represent ±SEM from three independent experiments, Welch's unpaired 2-tailed t test. Scale bar, 10 μm. (E) Western blot of MCL-1 after siRNA-mediated knockdown. At least three independent experiments were quantified. Error bars represent ±SD. (F) Working model of MCL-1 regulation of mitochondrial dynamics. See also Figure S4.

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