單分子定位顯微鏡揭示哺乳動物過渡區蛋白在分子解析度下的空間排列

Abstract

過渡區（TZ）位於初級纖毛基部（basal body）的特化區域，為控制和纖毛生成相關蛋白進出胞器的擴散屏障。到目前為止，在TZ中招募的許多蛋白質在空間排列上仍然不明確。單分子定位顯微技術（SMLM）之發展使生物學家能夠在奈米尺度下研究大分子結構。通過有效減少點擴散函數間的重疊使得定位精確度提升，單分子定位顯微技術突破光學顯微鏡的繞射極限並實現十至二十奈米的解析度。近年問世的擴展顯微技術使得超解析顯微的解析度獲得更進一步的提升。 本研究使用結合dSTORM（direct stochastic optical reconstruction microscopy）及U-ExM（ultra-expansion microscopy）的Ex-dSTORM同時在光學上及樣本上提升解析度，來建立哺乳類過渡區五種蛋白在軸向及徑向上的分布。我們發現CEP290在Ex-STORM的解析度下顯示了兩層在以前它被認為是厚的單層結構，而這可能與纖毛的生長狀態高度相關。基底層皆存在於纖毛生長之前的母中心粒和子中心粒上，遠端層伴隨著纖毛生長出現和其他TZ蛋白募集。在這裡，我們使用位於DAP的CEP164作為空間上的參考基準，描述了五個TZ蛋白的縱向和橫向分佈的覆蓋範圍。

As a specialized region located at the root of the cilium, transition zone serves as a diffusion barrier that regulates proteins associated with ciliopathy entry and exit from the organelle. So far, there are numerous proteins recruited in TZ remain ambiguous in the spatial arrangement. The development of single-molecule localization microscopy (SMLM) has enabled biologists to investigate substructures at the nanoscale. By effectively reducing the overlap between point spread functions to improve positioning accuracy, single-molecule localization microscopy surpasses the diffraction limit of conventional optical microscopy, enabling a resolution of ten to twenty nanometers. The expansion microscopy that came out in recent years has further improved the resolution of super-resolution microscopy. In this research, Ex-STORM, which is combined with dSTORM (direct stochastic optical reconstruction microscopy) and U-ExM (ultra-expansion microscopy), was utilized to improve the resolution optically and on the sample simultaneously. We found that CEP290 shows two layers under the resolution of Ex-STORM while it is thought to be one thick layer before. It could be highly related to the growing state of cilium. The basal layer exists on both mother and daughter centrioles before ciliogenesis, and the distal layer appears with the cilium growth and TZ proteins recruitment. Here, we depicted the coverage of five TZ proteins with CEP164, which belongs to the DAPs.