Single-molecule localization microscopy of expanded mammalian centrioles unravels the ultrastructural constitution of distal appendages.

Distal appendages (DAPs) are vital in ciliogenesis, mediating membrane docking and ciliary gating. Although the nine-fold symmetric constitution of DAP proteins has been investigated via superresolution microscopy analyses, the root structure of DAPs remains elusive owing to insufficient spatial optical resolution. Here, we proposed the in situ drift correction for optimized two-color imaging incorporating single-molecule localization microscopy with sample expansion approaches to achieve effective localization precision down to sub-5 nm. The robust workflow enables us to systematically perform the ultrastructural imaging study of protein mapping within cells, not limited to the proximity of coverslips. Notably, our findings unraveled the ultrastructural arrangement of the core DAP proteins (CEP83, CEP89, SCLT1, FBF1, and CEP164) and their spatial connection to microtubule triplets. Surprisingly, we found that C2CD3 was localized near a tubule of triplets guiding the DAP formation from microtubules to outer components of blades with nine-fold symmetry. We further identified the dual-layered distribution of ODF2. Intriguingly, the distal-layered ODF2 displayed a high spatial correlation with other DAP proteins, acting as a structural coordinator at the DAP root. Moreover, our results showed that the N- and C-terminus of ODF2 jointly encircled the microtubule triplets. Together, our studies suggest the unprecedented architectural framework of DAPs and provide a pragmatic roadmap for protein mapping with optical resolution at a molecular level.