多功奈米顯微術揭示哺乳類中心粒遠端附屬物之超微結構

Abstract

遠端附屬物在纖毛形成中至關重要，因為它們在早期纖毛發生過程中調控囊泡和纖毛與質膜的對接。許多遠端附屬物蛋白，其九重對稱排列，已通過超分辨率顯微鏡分析進行研究。然而，由於空間分辨率和資料產出率不足，要達到對從中心粒壁發展出的遠端附屬物結構的全面超微結構理解仍然具有挑戰性。為了提高蛋白質映射的空間分辨率，我們提出了一種實用的兩色Ex-dSTORM成像策略。我們的成像工作流程使我們能夠將光學顯微鏡的解析度極限推向分子級水平，從而能夠在完整細胞架構內以前所未有的分辨率解開遠端附屬物及其相關蛋白質的高階蛋白質結構。根據我們的成像結果，我們建立了一個超高解析度的遠端附屬物對中心粒的三維模型。此外，我們的研究發現表明ODF2在協調和維持遠端附屬物的九重對稱中起輔助作用。總的來說，我們制定了一個基於細胞器的漂移校正方法和一種最小串擾的兩色解決方案，可靠地促進了在水凝膠樣品複合體的深處進行擴展的遠端附屬物結構的定位顯微鏡成像。為了進一步提高資料產出率，我們提出了一種簡單的多目標單分子定位顯微術方法，稱為buffer-exchanged STORM（beSTORM），用於在單輪標記中可視化多個超高解析度蛋白質複合物。此方法利用不同緩衝條件下可區分的光子閃爍反應，引入了一個額外的維度，以區分單個分子，而不考慮其光譜特性。通過簡單的緩衝液交換，beSTORM實現了光譜無限制的多目標單分子定位顯微術成像，並且串擾極小。與擴展顯微鏡的直接結合顯示了在單一發射顏色中在分子級水平上解析多達六種蛋白質的能力，而不會出現色差。總的來說，beSTORM提供了一個高度兼容的成像方法之平台，為高度多通道奈米顯微術的發展提供了重要的一步，使其在生物系統中探索多個目標並有著奈米級精度。

Distal appendages (DAs) are vital in cilia formation, as they mediate vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Numerous DA proteins, which arrange in a nine-fold symmetry, have been studied using superresolution microscopy analyses. However, achieving an extensive ultrastructural understanding of the DA structure developing from the centriole wall remains challenging due to insufficient spatial resolution and throughput. To enhance the spatial resolution of protein mapping, we proposed a pragmatic imaging strategy for two-color Ex-dSTORM. Our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, allowing us to unravel the ultra-resolved higher-order protein complexes of the DA and its associated proteins with an unprecedented mapping resolution inside intact cells. Based on our imaging results, we constructed an ultra-resolved 3D model of the DA against the centriole. Moreover, our findings suggest that ODF2 plays an auxiliary role in coordinating and maintaining the nine-fold symmetry of DA. Collectively, we devised an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, facilitating robust localization microscopy imaging of expanded DA structures within the depths of gel-specimen composites. To further increase throughput, we proposed a simple multi-target SMLM approach called buffer-exchanged STORM (beSTORM), for visualizing multiple super-resolved protein complexes in a single round of labeling. This method leverages the distinguishable photo-blinking responses to distinct buffer conditions, introducing an additional dimension to differentiate between single molecules irrespective of their spectral properties. Through straightforward buffer exchanges, beSTORM achieves spectrum-unlimited multi-target SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color, without experiencing chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.