Single Molecule Tracking of Cilia Intraflagellar Transport

Abstract

Primary cilia are hair-like cellular organelles essential for cellular signaling. The structural and functional uniqueness and integrity of cilia rely on two critical processes: the transition zone (TZ) cargo sorting, a selective process regulating molecular traffic, and intraflagellar transport (IFT), a bidirectional system delivering cargo along the ciliary axoneme. Defects in TZ cargo sorting and IFT are common causes of ciliopathies. Understanding these processes is critical to revealing the fundamental cilia biology and disease mechanisms.

While structural approaches have been utilized to resolve the composition of TZ and IFT trains, they don’t provide the dynamic ciliary trafficking details. Complementarily, single particle tracking (SPT) methodology provides insights into live cell cilia transport dynamics, however, constrained by the resolution of microscopes (~200 nm), SPT lack the ability to resolve the nanoscale trajectories of individual trains and cargoes. Therefore, single-molecule or nanoscale-resolution methods are required to elucidate the instantaneous motions of these components. Recent super-resolution technique MINFLUX holds potential to track single fluorophores with nanometer spatial and millisecond temporal resolution, enabling direct visualization of detailed IFT train dynamics like TZ cargo sorting and microtubule switching.

Here, we leveraged MINFLUX microscopy to dissect the IFT process in live cells. First, we established the SPT model of IFT trains in primary cilia using mouse inner medullary collecting duct-3 (mIMCD3) cells stably expressing Halo-IFT88 (IFT-B core) and ARL13B-EGFP (ciliary marker). To minimize interference from nonspecific signals within the cell body, we used poly-D-lysine to induce cilia growth toward the coverslip and applied TIRF/HILO imaging. To enable single molecule imaging, we used a dual-labeling strategy (sparse JFX650 for single molecule tracking, saturated JF549 for ciliary context). This enabled direct visualization of IFT train trajectories, with measured anterograde/retrograde velocities consistent with reported values. Then, we benchmarked MINFLUX system by tracking Halo-tagged kinesin-II in U2OS cells, resolving 16 nm stepping events with <4 nm precision, validating its utility in crowded intracellular environments. This work lays the groundwork for future applications of MINFLUX in elucidating TZ cargo sorting and tracking IFT train microtubule switch events along primary cilia in live mammalian cells.

Date of Award	2025
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Zhen LIU (Supervisor)