Multiplexed nanoscopy unveils the ultrastructural constitution of mammalian centriole distal appendages

Ting-Jui Ben Chang^1,2,3,4*, T.Tony Yang^1,2

¹Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan
²Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan
³Department of Physics, National Taiwan University, Taipei, Taiwan
⁴Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica and National Taiwan University, Taipei, Taiwan

* Presenter:Ting-Jui Ben Chang, email:r04941038@g.ntu.edu.tw

Distal appendages (DAPs) are vital in cilia formation, as they mediate vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Numerous DAP proteins, which arrange in a nine-fold symmetry, have been studied using super-resolution microscopy analyses. However, achieving an extensive ultrastructural understanding of the DAP 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 DAP and its associated proteins with an unprecedented mapping resolution inside intact cell. Based on our imaging results, we constructed an ultra- resolved 3D model of the DAP against the centriole. Moreover, our findings suggest that ODF2 plays an auxiliary role in coordinating and maintaining the nine-fold symmetry of DAP. Collectively, we devised an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, facilitating robust localization microscopy imaging of expanded DAP 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.

Keywords: Super-resolution microscopy, Single-molecule localization microscopy, Expansion microscopy, Primary cilium, Distal appendage