Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha-subunit of F1-ATP synthase (ATPa), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPa is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria-rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria-less, endoplasmic reticulum (ER)-rich cortical region and the mitochondria-rich internal region. ATPa was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria-enriched fraction, a considerable amount of ATPa and F1-ATP synthase beta-subunit (ATPb) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1-ATP synthase gamma-subunit (ATPc) and F0-ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPa and ATPb. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.

The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.