Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Department of Biology, Technion-Israel Institute of Technology, 32000, Haifa, Israel.
Sci Rep. 2022 Jun 10;12(1):9609. doi: 10.1038/s41598-022-13547-w.
Successful gamete fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a live imaging-based adhesion molecule (LIAM) assay to study gamete adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell-cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gamete adhesion molecule pair, IZUMO1 and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO1 or JUNO was partly translocated to the opposite cells as discrete spots; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the LIAM assay, we investigated the species specificity of IZUMO1 and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO1 and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO1 and JUNO from different species.
成功的配子融合需要物种特异性的膜粘连。然而，通过低通量显微镜观察，很难实时研究配子中粘连分子的相互作用。因此，我们开发了一种基于活细胞成像的粘连分子（LIAM）测定法，以研究培养细胞中的配子粘连分子相互作用。首先，我们修改了先前为引入培养细胞的融合剂建立的融合测定法，并证实我们的活细胞成像技术可以在修改后的融合测定法中可视化细胞-细胞融合。接下来，我们用包括哺乳动物配子粘连分子对 IZUMO1 和 JUNO 在内的粘连分子代替融合剂，并检测它们在相邻细胞接触界面的时间积累。积累的 IZUMO1 或 JUNO 部分作为离散斑点转移到相反的细胞中；其相互作用所需的氨基酸突变会损害积累和转移。通过使用 LIAM 测定法，我们研究了不同物种的鼠、人、仓鼠和猪的 IZUMO1 和 JUNO 的物种特异性。在同种和一些种间组合中观察到 IZUMO1 和 JUNO 的积累和转移，这表明来自不同物种的 IZUMO1 和 JUNO 可能具有可互换的组合。
