Internalization of isolated functional mitochondria: involvement of macropinocytosis

Abstract

In eukaryotic cells, mitochondrial dysfunction is associated with a variety of human diseases. Delivery of exogenous functional mitochondria into damaged cells has been proposed as a mechanism of cell transplant and physiological repair for damaged tissue. We here demonstrated that isolated mitochondria can be transferred into homogeneic and xenogeneic cells by simple co-incubation using genetically labelled mitochondria, and elucidated the mechanism and the effect of direct mitochondrial transfer. Intracellular localization of exogenous mitochondria was confirmed by PCR, real-time PCR, live fluorescence imaging, three-dimensional reconstruction imaging, continuous time-lapse microscopic observation, flow cytometric analysis and immunoelectron microscopy. Isolated homogeneic mitochondria were transferred into human uterine endometrial gland-derived mesenchymal cells in a dose-dependent manner. Moreover, mitochondrial transfer rescued the mitochondrial respiratory function and improved the cellular viability in mitochondrial DNA-depleted cells and these effects lasted several days. Finally, we discovered that mitochondrial internalization involves macropinocytosis. In conclusion, these data support direct transfer of exogenous mitochondria as a promising approach for the treatment of various diseases.

Keywords: macropinocytosis; mitochondrial transfer; rho0 cells.

Fig. 1

(A) Mitochondria isolated from EMCs-DsRed2 mito cells. Phase contrast image (left), fluorescence image (middle) and merged image (right). Scale bars, 50 μm. (B) Transmission electron microscopy of isolated mitochondria. Inset: a high magnification image of isolated mitochondria; scale bar, 500 nm. (C) Characterization of isolated mitochondria by Zetasizer. Particle size (upper graph) and zeta potential distribution (lower graph) were measured. Av, average.

Fig. 2

(A) Live images of GFP-expressing H9c2 cells containing DsRed2-labelled human mitochondria. Phase contrast image (left) and merged fluorescent image (DsRed and GFP; right). White arrow heads: transferred DsRed2-mitochondria; scale bars, 100 μm. (B and C) Immunofluorescent staining of the H9c2 cells transferred with human mitochondria. Merged fluorescence images with human mitochondria (red) and DsRed2 protein (green; B, left), or human mitochondria (red) and the mitochondrial outer membrane receptor TOM20 (green; C, left). Nuclei were stained with DAPI (blue). The right panels in B and C show 3D reconstructed images of the left panels. Left panel inset: focused sites of 3D reconstructed images. White arrow heads indicate transferred mitochondria; scale bars, 100 μm. (D and E) PCR analysis of the H9c2 cells transferred with human mitochondria. (D) Human mtDNA detection from the PCR products, and (E) the relative content ratio of human mtDNA to human nuclear DNA in EMCs. Error bars represent SEM. **Significantly different, P < 0.01. EMCs, Human uterine endometrial gland-derived mesenchymal cells; H9c2, rat H9c2 cardiomyoblasts; H9c2-hmito, H9c2 cells transferred with human mitochondria; DDW, distilled deionized water; hN, human nuclear DNA; hmito, human mtDNA; rN, rat nuclear DNA; rM, rat mtDNA.

Fig. 3

(A) Representative live fluorescence image of EMCs transferred with DsRed2-labelled human mitochondria. White arrow heads indicate the transferred mitochondria; scale bar, 100 μm. (B) Immunoelectron microscopy of EMCs transferred with DsRed2-labelled human mitochondria. Inset: high magnification image of the transferred DsRed2-labelled mitochondria. Black arrow heads indicate DsRed protein; scale bar, 100 nm. (C–E) Dose-dependent increase in the percentage of DsRed-positive cells. (C) Representative live fluorescence images of transferred cells; scale bar, 200 μm. (D) Total number of cells and mitochondria-transferred cells using image-based cytometer analysis. Black and white bar sections indicate cells with and without exogenous mitochondria, respectively. (E) FACS analysis of mitochondria-transferred cells. Controls (cells without mitochondrial transfer) are represented by the black dots, and cells with mitochondrial transfer by the red dots Control, no mitochondria delivery; Mito 5–20, 5–20 μg/ml of mitochondria delivery. (F and G) Time course of transferred mitochondria. (F) Representative live fluorescence images and FACS analysis of transferred cells on day 1, 3 and 7 after mitochondrial transfer; scale bar, 100 μm. (G) Control (cells without mitochondrial transfer) are represented by the black dots, and cells with mitochondrial transfer by the red dots.

Fig. 4

Time-lapse fluorescent microscopy images of mitochondrial transfer. The numbers are time elapsed (h) since the initiation of co-incubation. The black and white arrows indicate the same mitochondria; scale bar, 100 μm. The movie file is supplemented in Video S1.

Fig. 5

Cell viability assay in mitochondria-transferred ρ0 cells on day 1, 3 and 7 after mitochondrial transfer. ρ0, no mitochondria delivery; Mito 2.5–25, 2.5–25 μg/ml of mitochondria delivery. Error bars represent standard error of the mean. *Significantly different, P < 0.05.

Fig. 6

Measurements of cellular bioenergetics after mitochondrial transfer. (A) Investigation of mitochondrial function in terms of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in mitochondria-transferred ρ0 cells on day 3 after mitochondrial transfer. Black arrows indicate the time of addition of each mitochondrial functional modifier. ρ0, no mitochondrial delivery; Mito 6–24, 6–24 μg/ml mitochondria delivery; EMCs, human uterine endometrial gland-derived mesenchymal cells. Data are expressed as mean ± standard error. (B) Basal OCR, maximal OCR, basal respiration, maximal respiration, adenosine triphosphate (ATP) production and spare respiratory capacity (spare capacity) of the cells. Error bars represent standard error. *Significantly different, P < 0.05.

Fig. 7

Impact of the macropinocytosis inhibitor EIPA on mitochondrial transfer. (A) Representative live fluorescence images and (B) FACS analysis for mitochondrial transfer performed in the presence of EIPA. Scale bar, 100 μm. (B) Cells without mitochondrial transfer are represented by the black dots and cells with mitochondrial transfer by the red dots. Control, no mitochondria delivery No EIPA, no EIPA treatment; EIPA 25, 25 μM EIPA treatment; EIPA 50, 50 μM EIPA treatment.