Differential roles of microtubules in the two formation stages of membrane nanotubes between human mesenchymal stem cells and neonatal mouse cardiomyocytes

Membrane nanotubes (MNTs) are a kind of novel way for communication between two distant cells. It was recently shown that MNTs can be formed between distressed cardiomyocytes (CMs) and mesenchymal stem cells (MSCs). As a cytoskeleton-containing structure, the role of microtubules in MNTs is not fully understood. Here, we investigated this question. By membrane dye staining, we found that the numbers of MNTs between human MSCs (hMSCs) and distressed neonatal mouse CMs (NMCMs) increased gradually from 3 to 16 h and remained constant from 16 to 30 h, which were identified as active formation stage (the 1st stage, ≤16 h in coculture), and mature and stable stage (the 2nd stage, >16 h in coculture), respectively. In the 1st stage, more MNTs originated from hMSCs, whereas more MNTs originated from NMCMs in the 2nd stage. The formation of MNTs was affected when microtubules were disrupted by nocodazole in the 1st stage, but not in the 2nd stage. MNTs became shorter and thinner when microtubules were disrupted in the 2nd stage. Immunofluorescence staining and flow cytometry showed that mitochondria in hMSCs were transported into distressed NMCMs, which was suppressed by nocodazole in the 2nd stage. Tunnel staining showed that hypoxia/reoxygenation-induced apoptosis of NMCMs only in the 2nd stage could be rescued by direct, but not indirect, coculture with hMSCs. This rescue function was weakened when the mitochondrial functions of cocultured hMSCs were disrupted by EtBr or microtubules in cocultures were disrupted by nocodazole. All these results suggested that there are two stages for MNT formation, and microtubules played differential roles in the two stages: During the 1st stage, microtubules were required for MNT formation, whereas during the 2nd stage, microtubules were related to the morphological features of MNTs and played a key role in anti-apoptosis of MNTs by mitochondrial transfer.