Single-Molecule Tracking Reveals Varying Transport Speed Of Ift88 Proteins At The Base Of Mammalian Primary Cilia

An essential trafficking mechanism of ciliogenesis is intraflagellar transport (IFT). The IFT processes at the ciliary base were largely unknown based on the diffraction-limited kymograph imaging. Here, we optimize single-molecule tracking localization microscopy to study IFT proteins at the ciliary base by observing IFT88-mEOS4b in live human retinal pigment epithelial cells. Surprisingly, we found that IFT88 proteins "switched gears" multiple times from ciliary base to cilium, revealing region-dependent slowdown of IFT proteins at the ciliary base: a slow to relatively fast movement from distal appendages (DAPs) to proximal transition zone (TZ), slow again in the distal TZ, and fastest in the ciliary compartment (CC). Our results further revealed that IFT88 could travel between the DAPs and the axoneme without following DAP structures.