Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Abstract

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.

Figure 1

Rescue of UV-treated PC12 cells in cocultures. (a) The level of cell death of UV-treated cells decreased in coculture condition. CTB-labelled PC12 cells were treated with UV light and grown in monoculture or in coculture with untreated cells for 24 h. The percentage of dead cells was quantified by staining with annexin V-AF488. UV-treated cells were also incubated for 24 h in the presence of 80 μM Z-VAD-FMK or in conditioned medium from untreated cells as indicated. (b) TNTs (arrows) were formed between CTG-labelled (CTG, green) and -unlabelled cells in control and UV treatment conditions but not in the presence of 350 nM cytoB (UV+cytoB). The cocultures were stained with WGA-AF594 (WGA, red) and imaged by confocal microscope. Scale bars, 20 μm. (c) Three hundred and fifty nanomolar cytoB abolished the formation of TNTs. The number of TNT between CTG-labelled cells (green circles), CTG-unlabelled cells (white circles) or CTG-labelled and -unlabelled cells were counted in the conditions as in (b). (d) Three hundred and fifty nanomolar cytoB inhibited the rescue effect. CTB-labelled cells treated with UV were cocultured with untreated cells (unlabelled) in the absence or presence of 350 nM cytoB for 24 h. The percentage of dead cells in both cell populations was quantified. NS, not significant; *P<0.05; **P<0.01

Figure 2

UV-treated PC12 cells form TNTs containing microtubules. (a) TNT formed by UV-treated cells (CTG-labelled, green) contained CTG (arrowhead) and microtubules (arrow). (Right) TNTs (arrow) formed by control cells did not contain CTG and microtubules. The cells were stained with WGA-AF633 (WGA, cyan) and immunostained with anti-α-tubulin antibody to visualize microtubules (red) and imaged by confocal microscope. (b and c) When the cocultures were treated with 10 μM Noc or 80 μM Z-VAD-FMK for 24 h, the CTG-TNTs (arrowheads) did not contain microtubules. (d) Statistical evaluation of frequencies of marker occurrence in TNTs between CTG-labelled and -unlabelled cells in the conditions as in (a–c). The ratio of TNTs containing microtubules but no CTG (grey bars), CTG but no microtubules (red bars) or microtubules and CTG (green bars) to total WGA-stained TNTs were calculated. Scale bars, 10 μm. **P<0.01

Figure 3

The characteristics of MT-TNTs. (a) The colocalization of microtubules (arrowhead) and F-actin (arrow) in a TNT between UV-treated (CTB-labelled, blue) and untreated cells. F-actin was stained with phalloidin-AF488. Microtubule was immunostained with anti-α-tubulin antibody and imaged by confocal microscope. (b) CTG-TNT (arrow) contained detyrosinated α-tubulin (arrowhead). CTG-labelled cells (CTG, green) were treated with UV and cocultured with untreated cells for 24 h. Cells were then immunostained with anti-detyrosinated α-tubulin (red) and imaged by confocal microscope. (c) EB3-mCherry (arrowheads) moved towards the end of a CTG-TNT (arrow). EB3-mCherry-transfected cells were labelled with CTG, treated with UV and cocultured with untreated cells for 24 h. Cells were subjected to time-lapse confocal imaging. (d) The membrane fluidity of CTG-TNTs was less than normal TNTs. The half-time of recovery (t_1/2) of fluorescence of WGA-AF594 of TNTs between control cells (control, n=15) and CTG-TNTs between UV-treated and -untreated cells (UV, n=13) was measured by FRAP. Scale bars, 10 μm. **P<0.01

Figure 4

Cells in the early stages of apoptosis form MT-TNTs. (a and b) MT-TNT- (arrows) connected UV-treated cells (UV) showed neither PS exposure (a) nor caspase-3 activity (b). CTB-labelled cells (CTB, blue) were treated with UV and then cocultured with untreated cells for 24 h. The cells were stained with annexin V-AF488 (PS, green) or the Image-iT LIVE Green Caspase-3/7 Detection Kit (Caspase-3/7, green), immunostained with anti-α-tubulin antibody (red) and imaged by confocal microscope. Note that dead cells (arrowheads) showed PS exposure (a) and caspase-3/7 activity (b) as positive control. (c) MT-TNT- (arrow) connected UV-treated cell (UV) contained less cyt c (arrowhead) than untreated cells. CTG-labelled cells (CTG, green) were treated with UV and then cocultured with untreated cells for 24 h. The cells were immunostained with anti-α-tubulin (red) and anti-cytochrome c (cyt c, cyan), and imaged by confocal microscope. (d) UV-treated cells formed MT-TNTs when cells lose cyt c. The RFI of anti-cyt c in untreated control cells (n=276), UV-treated cells without MT-TNT (n=202) and UV-treated cells connected with MT-TNT (n=67) was calculated as described in the Materials and Methods section. (e) The frequency distribution of RFI values of anti-cyt c in microtubule-containing and -deficient CTG-TNT- connected UV-treated cells. CTG-labelled cells were treated with UV and then cocultured with untreated cells in the absence or presence of 80 μM Z-VAD-FMK for 24 h. UV-treated cells connected with microtubule-containing CTG-TNT (n=67) or connected with microtubule-deficient CTG-TNT (Z-VAD-FMK treated, n=30) were imaged by confocal microscope and analysed as in (d). Scale bars, 10 μm. *P<0.05; **P<0.01

Figure 5

The presence and transport of mitochondria in CTG-TNTs. (a) Intact mitochondria (arrowheads) localized in a CTG-TNT (arrow). CTG-labelled cells (CTG, green) were treated with UV, cocultured with untreated cells for 18 h. Cells were stained with TMRM (red) and WGA-AF633 (WGA, cyan) and imaged by confocal microscope. (b) Mitochondria (arrowheads) translocated along a TNT towards the UV-treated cell. CTG-labelled cells (green) were treated with UV and cocultured with untreated cells for 18 h. Then, cells were stained with TMRM (red) and subjected to time-lapse confocal imaging. (c) Mitochondria (arrowhead) colocalized with microtubules (arrow) along a CTG-TNT. CTG-labelled cells (CTG, green) were treated with UV and cocultured with untreated cells for 24 h. The coculture was subsequently stained with MTDR (cyan), then immunostained with an anti-α-tubulin antibody (red) and imaged by confocal microscope. Scale bars, 10 μm

Figure 6

The rescue effect depends on the transfer of functional mitochondria. (a) The presence of DsRed2-mito (arrows) of healthy cells in a UV-treated cell. CTG-labelled cells (green) were treated with UV, cocultured with untreated cells expressing DsRed2-mito (arrowhead) for 24 h and imaged by confocal microscope. (b) The transfer of DsRed2-mito from healthy cells to UV-treated cells was inhibited by cytoB. The percentage of CTG-labelled cells that contained DsRed2-mito was calculated in control (without UV treatment) and in UV treatment in the absence or presence of 350 nM cytoB. (c) The ρ⁰ cells were less able to rescue the UV-treated cells than the normal cells (ρ⁺). Unlabelled ρ⁰ cells were cocultured with CTB-labelled UV-treated cells for 24 h. (Left) The ρ⁰ cells contained no mtDNA, whereas the ρ⁺ cells did (arrowheads). The ρ⁺ and ρ⁰ cells were stained with the Click-iT EdU Microplate Assay kit (Amplex UR, red) to detect EdU-labelled DNA and imaged by confocal microscope. Note that the nuclei (n) were strongly stained in both ρ⁺ and ρ⁰ cells. (d) The ρ⁰ and ρ⁺ cells formed comparable numbers of TNTs. TNTs formed in ρ⁰ and ρ⁺ cells were quantified under conditions with or without UV treatment (control). Scale bars, 10 μm. NS, not significant; *P<0.05; **P<0.01