Intraflagellar transport proteins undergo nonaxonemal staged hindrance between the recruiting distal appendages and the cilium

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which first requires IFT-protein recruitment, IFT-protein-motor-protein assembly, axonemal engagement of IFT-protein complexes, and transition zone (TZ) gating. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is exceedingly challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy (sptPALM), balancing the imaging requirements of tracking speed, tracking duration, and localization precision for IFT88-mEOS4b in live human retinal pigment epithelial (hTERT-RPE-1) cells. Intriguingly, we found that mobile IFT proteins "switched gears" multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal TZ, slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures, and reached the space enveloped by the ciliary pocket in the proximal TZ. Together, our live-cell superresolution imaging revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliogenesis homeostasis.