Superresolution Microscopy Reveals Staggered Arrangement of Mammalian Distal Appendages

Distal appendages play essential roles in ciliary membrane anchoring and ciliary protein recruitment. Previously we have revealed a hierarchical relationship among distal appendage proteins, including CEP83, CEP89, SCLT1, CEP164, and FBF1. Despite their importance, their relative placement on distal appendages remains largely unknown due to the small length scale beyond the diffraction limit. Using dual-color direct stochastic optical reconstruction microscopy (dSTORM), here we revealed a staggered arrangement of CEP83, CEP89, SCLT1, CEP164, and FBF1, with surprising alternating arrangement of CEP164 and FBF1. We also found the cell cycle-dependent distribution of TTBK2 on distal appendages, illustrating the coupling between cell cycle and ciliogenesis. Together, we have determined the hierarchical architecture of the distal appendages, suggesting their potential membrane interaction site and kinase recruiting site.