Perinuclear mitochondrial clustering for mesenchymal-to-epithelial transition in pluripotency induction

Abstract

Remodeled mitochondria are characteristic of pluripotent stem cells. However, a role for mitochondrial movement and distribution in pluripotency remains unknown. Here, we show that mitochondrial retrograde transport-mediated perinuclear clustering via dynein complex occurs at the early phase of pluripotency induction. Interestingly, this mitochondrial redistribution is regulated by Yamanaka factor OCT4 but not SOX2 or KLF4. This mitochondrial redistribution, which has effect on the efficiency of somatic cell reprogramming, also depends on DRP1-mediated mitochondrial fission. Importantly, perinuclear mitochondrial clustering is required for mesenchymal-to-epithelial transition (MET), an early step in reprogramming, during which β-catenin regulates the MET process. Furthermore, sufficient amount of β-catenin plays a key role in maintaining stabilization of E-CADHERIN. Taken together, these studies show that perinuclear mitochondrial clustering is an essential organellar step for MET process of pluripotency induction, which may shed light on the subcellular relationship between mitochondrial dynamics, pluripotency, and cellular morphology.

Keywords: Drp1; Dynein; Oct4; Wnt signaling; mesenchymal-to-epithelial transition.

Graphical abstract

Figure 1

Perinuclear mitochondrial clustering appears at the early phase of the somatic cell reprogramming (A) Representative images for mitochondrial distribution in MEFs transduced with FLAG or SKO (mtDsRed is shown in red, ACTIN-GFP in green, DAPI in blue) Scale bars: 10 μm. (B) Relative fluorescence intensity of the mitochondria in the perinuclear or periphery area indicated in MEFs transduced with FLAG or SKO at day 3 (n = 3 independent experiments). (C) Gene ontology analysis of the DEGs in MEFs transduced with FLAG and D3, D5, and D7 SKO quantified by RNA-seq. (D) Transcriptome dynamics obtained by K-means clustering (k = 8) of DEGs in MEFs transduced with FLAG or SKO based on their expression. (E) Clusters of upregulated and downregulated genes associated with dynein complex during reprogramming. Black line indicates dynamic expression pattern of each cluster. Levels of gene expression were represented along the y axis, and the days of reprogramming were represented along the x axis. The two subclusters about the dynein complex were obtained by K-means algorithm (Fujikura et al. 2021). (F) Protein-protein interaction (PPI) networks of key protein from dynein complex. (G) mRNA level of indicated genes in MEFs transduced with SKO (n = 5 independent experiments). Data are shown as mean ± standard deviations (SDs). p values were calculated using the Student’s t test. ^∗p < 0.05, ^∗∗∗p < 0.001. See also Figure S1.

Figure 2

Retrograde transport of mitochondria is essential for somatic cell reprogramming in a MET-dependent manner (A) Indicative images for mitochondrial distribution in MEFs transduced with SKO plus shRNA. Scale bars: 10 μm. (B) Reprogramming efficiency (represented by GFP-positive colonies) in MEFs transduced with SKO plus shRNA (n = 3 independent experiments). (C) Indicative images for mitochondrial distribution in MEFs transduced with SKO plus shRNA. Scale bars: 10 μm. (D) Reprogramming efficiency in MEFs transduced with SKO plus shRNA (n = 3 independent experiments). (E and F) Western blot analysis showing expression of E-CADHERIN in MEFs transduced with SKO plus shRNA at the indicated days (n = 4 independent experiments). Data are shown as mean ± SD. p values were calculated using the Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01 ^∗∗∗p < 0.001. See also Figure S3.

Figure 3

Wnt/β-catenin is essential for MET during somatic cell reprogramming (A) Western blot analysis showing expression of E-CADHERIN in MEFs transduced with SKO plus treatment at the indicated days (n = 4 independent experiments). (B) Western blot analysis of E-CADHERIN in MEFs transduced with SKO plus shRNA (n = 3 independent experiments). (C) Western blot analysis of E-CADHERIN in MEFs transduced with SKO plus shRNA together with CHIR at the indicated days (n = 3 independent experiments). (D) Western blot analysis of E-CADHERIN in MEFs transduced with SKO plus shRNA together with CHIR at the indicated days (n = 3 independent experiments). (E) Reprogramming efficiency (represented by GFP-positive colonies) in MEFs transduced with SKO plus shRNA, together with DMSO or CHIR treatment (n = 3 independent experiments). Data are shown as mean ± SD. p values were calculated using the Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01 ^∗∗∗p < 0.001. See also Figure S4.

Figure 4

The downregulation of E-CADHERIN induced by Dync1h1 silencing was accompanied by the decrease of β-catenin (A) Representative images for the distribution of E-CADHERIN and β-catenin in MEFs transduced with SKO plus shRNA (cells were stained with anti- E-CADHERIN and shown in red, β-catenin in green, and DAPI in blue). Scale bars: 10 μm. (B) Western blot analysis for total β-catenin in the cytoplasmic fraction versus the nuclear fraction of MEFs transduced with SKO plus shRNA. Mouse TUBULIN was used as a cytoplasmic control while H3 was used as a nuclear control (n = 3 independent experiments). (C) Schematic illustration of perinuclear mitochondrial clustering regulation of somatic cell reprogramming. Data are shown as mean ± SD. p values were calculated using the Student’s t test. ^∗∗p < 0.01 ^∗∗∗p < 0.001.