Subdiffraction Imaging Reveals Molecular Architecture at the Transition Zone of Primary Cilia

Recent studies have identified important regulating elements for transition zone (TZ) gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation mechanisms. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a custom-built stimulated emission depletion (STED) superresolution microscope, we revealed relative localizations of TZ proteins, IFT proteins, transition fiber (TF) proteins, and centriole proteins. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227±18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205±20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165±8 nm diameter. Surprisingly, CEP290 (antibody against C-terminal amino acids) was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. IFT88 was also surprisingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base. We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open.