Single particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia.

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT) involving in IFT-protein recruitment, axonemal engagement of IFT-protein complexes, etc. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is 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) for IFT88-mEOS4b in live human retinal pigment epithelial 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. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, suggesting it as a potential assembly site. Together, our live-cell single particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis. Movie S1 Movie S1 Representative time-lapse movie during a streaming sptPALM acquisition of stochastically activated IFT88-mEos4b detected along a primary cilium of a living cell. Each emitting event gave a short trajectory lasting several to tens of time points at 30 ms per frame. Bar, 1 μm. Movie S2 Movie S2 Three example trajectories of IFT88-mEos4b on one primary cilium illustrating possible movement of IFT88 at the ciliary base, overlaid on the static PALM image of IFT88 to visualize the contour of the cilium. Trajectories are color-coded based on recorded time points. Each frame was recorded at a 30-ms interval. Movie S3 Movie S3 Zoomed-in trajectory of an IFT88-mEos4b molecule (the one in the lower part of Movie S2) showing its movement in the transverse direction from the periphery to the center of the ciliary base. Movie S4 Movie S4 Zoomed-in trajectory of an IFT88-mEos4b molecule (the one in the middle of Movie S2) showing its confined localization. Movie S5 Movie S5 Zoomed-in trajectory of an IFT88-mEos4b molecule (the one in the upper part of Movie S2) showing its movement first traveling toward one direction and then changing to another.