Molecular resolution imaging reveals step-wise reduction of axonemal diameter and ultrastructural architecture of intraflagellar transport proteins.

Abstract

Primary cilia are microtubule-based organelles essential for mediating mechano-/photo-/chemo-sensations in mammalian cells. Intraflagellar transport (IFT) proteins are crucial components to support ciliary assembly, maintenance, and cellular signaling. The ciliary microtubule doublets of the ‘9 + 0’ arrangement serve as tracks for molecular motor-based transport along cilia. Although the tapering structure of primary cilia has been examined by electron microscopy, the spatial correlation between the IFT pattern and axonemal structure remains elusive due to insufficient light resolution. Here, we integrated single-molecule localization microscopy and sample expansion techniques achieving two-color imaging with effective localization precision of sub-5 nm to explore the axonemal ultrastructure and molecular organization of IFT and IFT-associated proteins in retinal pigment epithelial cilia. Our results revealed the step-wise reduction of ciliary diameter, different from the well-known paradigm showing a gradual change in the width of axonemal bundles. Surprisingly, our data showed that the IFT proteins congested near the end of the microtubule doubles within the ciliary compartment. In addition, we resolved the molecular architecture of IFT complexes, cargo, and motor proteins to characterize their relative localizations. We found that IFT proteins presented distinct distributions, including isolated units, paired configurations, or alternating arrangements in IFT trains. Together, our ultrastructural light nanoscopic imaging enables us to perform morphological analysis of primary cilia, which guides the localization of IFT complexes at segmented sites of cilia, suggesting the asynchronous axonemal growth of microtubule doubles.