Frolikova Michaela, Sur Vishma Pratap, Novotny Ivan, Blazikova Michaela, Vondrakova Jana, Simonik Ondrej, Ded Lukas, Valaskova Eliska, Koptasikova Lenka, Benda Ales, Postlerova Pavla, Horvath Ondrej, Komrskova Katerina
Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia.
Light Microscopy Core Facility, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czechia.
Front Cell Dev Biol. 2023 Aug 10;11:1110681. doi: 10.3389/fcell.2023.1110681. eCollection 2023.
Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual , and the membrane of -free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or -free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.
已知在卵细胞膜中表达的Juno蛋白和CD9蛋白对于精子与卵母细胞的结合和融合至关重要。尽管有证据表明这两种蛋白存在协同作用，但它们之间的相互作用尚未得到证实。在此，我们展示了使用3D STED超分辨率技术捕获的小鼠中期II期卵母细胞表面Juno蛋白和CD9蛋白的相互定位情况。在所检测蛋白的精确定位在卵细胞膜的不同区域得以确定，如微绒毛膜、单个微绒毛之间的平面膜以及无微绒毛区域的膜。通过透射电子显微镜和扫描电子显微镜图像分析证实了Juno蛋白和CD9蛋白定位的差异，结果显示所选膜区域之间蛋白质的存在情况存在显著差异。基于皮尔逊相关系数的超分辨率图像共定位分析支持了Juno蛋白和CD9蛋白在卵细胞膜中相互位置的证据，这一证据通过邻近连接分析得以确定。重要的是，在HEK293T/17转染细胞系中通过共免疫沉淀和质谱检测到了Juno蛋白和CD9蛋白之间的相互作用。为了更好地理解实验数据，通过比较同源建模制备了小鼠Juno蛋白和CD9蛋白的3D结构，并使用ClusPro服务器进行了多次蛋白质 - 蛋白质柔性侧链对接。通过分子动力学（MD）模拟在原子水平实时研究了蛋白质的动态状态。对接和MD模拟预测了Juno - CD9蛋白之间的相互作用和稳定性，也暗示了一种相互作用机制。使用多尺度方法，我们检测到在微绒毛卵细胞膜内Juno蛋白和CD9蛋白紧密相邻，但在平面膜或无微绒毛区域则不然。我们的研究结果表明，在精子附着之前，小鼠卵细胞膜蛋白网络中存在尚未明确的Juno蛋白和CD9蛋白相互作用。这些结果表明，Juno蛋白和CD9蛋白的相互作用网络可能有助于Juno蛋白首先与精子Izumo1结合，这是随后配子膜融合的前提条件。
