Research ArticleCELL BIOLOGY

Endoplasmic reticulum mediates mitochondrial transfer within the osteocyte dendritic network

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Science Advances  20 Nov 2019:
Vol. 5, no. 11, eaaw7215
DOI: 10.1126/sciadv.aaw7215
  • Fig. 1 Mitochondria distributed within the osteocyte dendritic network.

    (A) Flowchart of imaging ex vivo cultured primary osteocytes. (B) Confocal analyses of primary osteocytes from ex vivo cultured calvariae show the distribution of TOM20-labeled mitochondria in dendrites of phalloidin-labeled primary osteocytes. (C) Confocal images of primary osteocytes from cortical bone of 4-, 12-, and 18-month-old mice femurs. (D) Quantitative analysis of mitochondrial distribution in the osteocyte dendritic network with age. Kruskal-Wallis test with Dunn’s post hoc. Three independent experiments were conducted. At least three fields and 30 cells in each group were analyzed. Data are presented as means ± SEM. (E) SIM images of the interconnected dendritic processes of MLO-Y4 cells show the association of mitochondria (MTO) in tubulin along dendrites of osteocytes. (F and G) Confocal images and fluorescence intensity analysis show the association of MitoTracker Red (MTR)–labeled mitochondria, EdU (5-ethynyl-2′-deoxyuridine)–labeled mtDNA, and Alexa Fluor 647–labeled ATP. Scale bars, 10 μm. *P < 0.05, **P < 0.01.

  • Fig. 2 Mitochondrial transfer within the osteocyte dendritic process.

    (A) Dynamic movement of MTO-labeled mitochondria with GFP-labeled dendritic tubulin in MLO-Y4 cells using confocal live-cell images. (B) PKH26-labeled recipient MLO-Y4 cells cocultured with pTaqYFP-mito–transfected donor MLO-Y4 cells. (C) Time-lapse confocal images show dendritic mitochondria labeled with pTaqYFP-mito moving dynamically toward the adjacent PKH26-labeled MLO-Y4 cell. Inserted images show the contacts between pTaqYFP-mito–labeled donor mitochondria and PKH26-labeled recipient cell membrane. (D) Kymograph analysis shows pTaqYFP-mito–labeled donor mitochondria moving toward PKH26-labeled recipient cell. (E) Quantitative assessment of mitochondrial movement in dendrites. Data are presented as means ± SEM. (F) Cocultures of MLO-Y4 cells transfected with pTaqYFP-mito or cortactin-RFP, respectively. Transferred mitochondria from donor cell (enlarged images in insert I) and transferred mitochondria from recipient cell (enlarged images in insert II) shown via differential interference contrast (DIC)–labeled dendrites. (G) 3D confocal images of replated cocultured MLO-Y4 cells show that the transferred pTaqYFP-mito–labeled mitochondria are present inside the cortactin-RFP–labeled recipient cell. Scale bars, 10 μm.

  • Fig. 3 Mitochondria transfer via the primary osteocyte dendritic network.

    (A) A cre-dependent mito-Dendra2 cassette was inserted into the Rosa26 locus (schematic 1: black arrowheads, loxP sites; stop symbol, termination cassette). When crossed to Dmp1Cre mouse line, the termination signal is excised to produce the PhAM line with osteocyte-specific labeling of mitochondria (schematic 2). Irradiating mito-Dendra2 (green) with a 405-nm laser results in a photoswitch to mito-Dendra2 (red) (schematic 3). (B) Calvariae extracted from young Dmp1CrePhAMfloxed mice and irradiated by 405-nm laser in area a, but not in area b, are imaged after 2 days ex vivo culture. (C) Area a and area b before and after photoswitch. (D) Confocal images of photoswitched primary osteocytes in situ, after ex vivo culture, demonstrate the transfer of mito-Dendra2–labeled mitochondria (magenta) to the recipient osteocytes with mito-Dendra2–labeled mitochondria (green). (E) Schematic of photoswitching primary osteocytes and (F) confocal images of primary osteocytes isolated from Dmp1CrePhAMfloxed mice photoswitched in area a. (G) Photoswitched and non-photoswitched primary osteocytes were replated after coculture, and confocal images show the transfer of mitochondria between primary osteocytes. (H) Quantitative analysis shows the ratio of cells with transferred mito-Dendra2 (magenta) mitochondria at 12-, 18-, 24-, and 48-hour coculture. One-way analysis of variance (ANOVA) with Dunnett’s post hoc. Three independent experiments were conducted. Data are presented as means ± SEM. (I) Number of transferred Dendra2–labeled mitochondria (magenta) in each cell increases over time in culture. Kruskal-Wallis test with Dunn’s post hoc. Three independent experiments were conducted. At least 40 cells in each group were analyzed. Data are presented as means ± SEM. Scale bars, 10 μm. N.S., not significant; *P < 0.05, **P < 0.01.

  • Fig. 4 Healthy osteocytes rescue MLO-Y4 ρ° cells via dendritic connection through the transwell membrane.

    (A) Confocal images of the transwell membrane and its pores. (B) Dendritic processes of MLO-Y4 cells extend through the pores of the membrane and connect with cocultured MLO-Y4 cells. (C) qPCR results show the depletion of mitochondrial genes Mt-ATP6/8 and Mt-CO3 in MLO-Y4 ρ° cells. (D) Mito-Dendra2 green–labeled mitochondria from primary osteocytes transfer through the pores of the transwell membrane. (E) 3D confocal images of transwell membrane coculture show the transfer of mitochondria from mito-Dendra2 green–labeled primary osteocytes to MLO-Y4 ρ° cells (ρ° cells). Left: Transverse view of 3D cocultured primary osteocyte and ρ° cells (TM, transwell membrane). Right: View of ρ° cells from above. (F) T1: ρ° cells cocultured with ρ° cells in the transwell culture system; T2: ρ° cells cocultured with healthy MLO-Y4 cells (parental cells); T3: ρ° cells cocultured with primary osteocytes. (G) Quantitative analysis of 3D confocal images shows that the healthy osteocytes are able to rescue ATP production (labeled by Alexa Fluor 647) in ρ° cells. Kruskal-Wallis test with Dunn’s post hoc. Three independent experiments were conducted. At least 30 cells in each group were analyzed. Data are presented as means ± SEM. Scale bars, 10 μm. **P < 0.01, ***P < 0.001.

  • Fig. 5 ER facilitates mitochondrial transfer between osteocytes.

    (A and B) 3D confocal analysis and fluorescence intensity analysis of primary osteocytes from ex vivo cultured calvariae. Images show the colocalization of TOM20-labeled mitochondria and calnexin-labeled ER in phalloidin-labeled dendrites. (C) Confocal time-lapse images of live MLO-Y4 cell show the colocalization of pTagYFP-mito–labeled mitochondria and RFP-labeled ER during transfer from the cytoplasm to the dendritic process. (D and E) 3D image and fluorescence intensity analysis show the colocalization between MTR-labeled mitochondria and GFP-labeled ER. (F) Fluorescence intensity analysis of dendritic processes shows the colocalization between GFP-labeled ER and MTR-labeled mitochondria. (G) Confocal time-lapse images show that GFP-labeled ER is dynamically colocalized with MTR-labeled mitochondria during movement toward the adjacent MLO-Y4 cell. (H and I) Kymograph and overlap and Pearson’s coefficient analyses show the dynamic association of GFP-labeled ER and MTR-labeled mitochondria. Scale bars, 10 μm.

  • Fig. 6 Mfn2 regulates mitochondrial transfer between osteocytes.

    (A) YFP-labeled Mfn2 and MTR-labeled mitochondria are colocalized in the MLO-Y4 cell dendrite. (B and C) Colocalization of YFP-labeled Mfn2 and MTR-labeled mitochondria during movement toward adjacent MLO-Y4 cells. (D) Dynamic mitochondrial movement was impeded by knockdown of Mfn2. (E) Knockdown of Mfn2 attenuates movement of the pTaqYFP-mito–labeled mitochondria in dendrites. Two-tailed Student’s t test. Three independent experiments were conducted. At least 10 cells in each group were analyzed. Data presented as means ± SEM. (F and G) Mfn2 knockdown significantly reduces distribution of pTaqYFP-mito–labeled mitochondria within dendrites of MLO-Y4 cells. Kruskal-Wallis test with Dunn’s post hoc. Three independent experiments were conducted. At least 30 cells in each group were analyzed. Data presented as means ± SEM. (H and I) T1: MLO-Y4 ρ° cells (ρ° cells) cocultured with ρ° cells in the transwell culture system; T2: ρ° cells cocultured with healthy MLO-Y4 cells (parental cells); T3: ρ° cells cocultured with Mfn2 knockdown MLO-Y4 cells (si-Mfn2 cells). Healthy cells rescue ATP production (labeled by Alexa Fluor 647) in ρ° cells but not in cells lacking Mfn2. Kruskal-Wallis test with Dunn’s post hoc. Three independent experiments were conducted. At least 30 cells in each group were analyzed. Data presented as means ± SEM. (J and K) Protein level of Mfn2 in cortical bone decreases with age. One-way ANOVA with Dunnett’s multiple comparisons test. Data presented as means ± SEM. Scale bars, 10 μm. *P < 0.05, **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaaw7215/DC1

    Fig. S1. No mitochondrial transfer from conditioned medium to MLO-Y4 cells.

    Fig. S2. Osteocytes do not pass through the pores of the transwell membrane.

    Fig. S3. Depleted mtDNA and impeded ATP production in MLO-Y4 ρ° cells.

    Fig. S4. Parental MLO-Y4 cells and primary osteocytes can transfer mitochondria to MLO-Y4 ρ° cells.

    Fig. S5. Mitochondrial distribution and ER movement in osteocyte dendrites.

    Fig. S6. Knockdown of Mfn2 has no effect on ATP production and the level of mtDNA.

    Fig. S7. Mfn2 knockdown impedes mitochondrial transfer in MLO-Y4 cells.

    Fig. S8. VAPB knockdown impedes mitochondrial transfer in MLO-Y4 cells.

    Fig. S9. Hypothetical model of ER-mediated mitochondrial transfer.

    Table S1. Primer sequences for qPCR.

    Movie S1. pTaqYFP-mito–labeled mitochondria (green) in the dendrite dynamically migrate toward the PKH26-stained cells (magenta).

    Movie S2. pTaqYFP-mito–labeled mitochondria (green) dynamically move from the cytoplasm to the dendritic process and are associated with RFP-labeled ER (magenta).

    Movie S3. Transfer of MTR-labeled mitochondria (magenta) within dendrites is dynamically colocalized with GFP-labeled ER (green).

    Movie S4. Transfer of MTR-labeled mitochondria (magenta) is dynamically colocalized with YFP-labeled Mfn2 (green) within dendrites.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. No mitochondrial transfer from conditioned medium to MLO-Y4 cells.
    • Fig. S2. Osteocytes do not pass through the pores of the transwell membrane.
    • Fig. S3. Depleted mtDNA and impeded ATP production in MLO-Y4 ρ° cells.
    • Fig. S4. Parental MLO-Y4 cells and primary osteocytes can transfer mitochondria to MLO-Y4 ρ° cells.
    • Fig. S5. Mitochondrial distribution and ER movement in osteocyte dendrites.
    • Fig. S6. Knockdown of Mfn2 has no effect on ATP production and the level of mtDNA.
    • Fig. S7. Mfn2 knockdown impedes mitochondrial transfer in MLO-Y4 cells.
    • Fig. S8. VAPB knockdown impedes mitochondrial transfer in MLO-Y4 cells.
    • Fig. S9. Hypothetical model of ER-mediated mitochondrial transfer.
    • Table S1. Primer sequences for qPCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). pTaqYFP-mito–labeled mitochondria (green) in the dendrite dynamically migrate toward the PKH26-stained cells (magenta).
    • Movie S2 (.mp4 format). pTaqYFP-mito–labeled mitochondria (green) dynamically move from the cytoplasm to the dendritic process and are associated with RFP-labeled ER (magenta).
    • Movie S3 (.mp4 format). Transfer of MTR-labeled mitochondria (magenta) within dendrites is dynamically colocalized with GFP-labeled ER (green).
    • Movie S4 (.mp4 format). Transfer of MTR-labeled mitochondria (magenta) is dynamically colocalized with YFP-labeled Mfn2 (green) within dendrites.

    Files in this Data Supplement:

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