Division of Biofunctional Sciences, Department of Integrated Health Sciences, Graduate School of Medicine, Nagoya University.
Division of Morphological Sciences, Kagoshima University Graduate School of Medical and Dental Sciences.
Biomed Res. 2022;43(4):107-114. doi: 10.2220/biomedres.43.107.
A live assay tool has been established to uncover the precise molecular mechanisms underlying complex cell fusion events in myoblasts. The novel cell-based assay, HiMy (HiBiT-based myoblast fusion), utilizes a recently developed split-luciferase technology. The assay successfully detected cell fusion in differentiating C2C12 myoblast cultures. This allowed us to measure mixing of the cytoplasm, which occurred several hours after the initiation of C2C12 differentiation. Unlike what was reported earlier, the fusion was detected a few hours after the initiation of differentiation. Thus, this assay is sensitive enough to monitor fusion events before they become detectable using conventional methods. Furthermore, a panel of laboratory compounds, including a variety of inhibitors of cellular enzymes or activities, were assayed using the HiMy assay. Lovastatin, a cholesterol biogenesis inhibitor, decreased HiMy activity by approximately 50%. In contrast, mevalonolactone, a precursor for cholesterol synthesis, increased fusion activity. These results confirmed the previous finding that the amount of cellular cholesterol positively correlates with the rate of myoblast fusion during myogenesis. These results indicate that the novel cell fusion assay is a quick, accurate, and robust method to monitor intercellular fusion events.
已建立一种活细胞检测工具，以揭示成肌细胞中复杂细胞融合事件背后的确切分子机制。新型基于细胞的测定法（HiMy，基于 HiBiT 的成肌细胞融合）利用了最近开发的分割萤光素酶技术。该测定法成功检测到分化的 C2C12 成肌细胞培养物中的细胞融合。这使我们能够测量细胞质的混合，细胞质混合发生在 C2C12 分化开始后的几个小时。与之前报道的不同，融合在分化开始后几个小时被检测到。因此，该测定法足够灵敏，可在使用常规方法检测到融合事件之前进行监测。此外，使用 HiMy 测定法对包括各种细胞酶或活性抑制剂在内的实验室化合物进行了检测。洛伐他汀是一种胆固醇生物合成抑制剂，可使 HiMy 活性降低约 50%。相比之下，鲨烯醇是胆固醇合成的前体，可增加融合活性。这些结果证实了之前的发现，即细胞胆固醇的量与成肌细胞融合过程中的成肌细胞融合率呈正相关。这些结果表明，新型细胞融合测定法是一种快速、准确和稳健的方法，可监测细胞间融合事件。
