Single-molecule localization microscopy reveals the ultrastructural root constitution of distal appendages in expanded mammalian centrioles

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP root structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of swellable mammalian DAP proteins. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to an electron microscopy level, thus achieving an unprecedented λ/200 mapping precision inside intact cells. Upon this workflow, we unravel the ultraresolved higher-order protein complexes of the core DAP. Intriguingly, C2CD3, microtubule triplet, and ODF2 jointly constitute the spatial basis of DAP, suggesting a unique configuration of the DAP assembly. Moreover, our results show that the distal-layered ODF2 labeled at the N- and C-terminus construct a fastening unit encircling the microtubule triplets. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded cellular structures deep into the gel-specimen composites.