Abstract
The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet–Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.
Highlights
Cilia are microtubule-based, finger-like cell protrusions which can be roughly divided into two categories: the motile cilia whose primary function is cell motility or translocation of materials along the surface of the ciliated epithelial cell[1] and the sensory cilia which house a variety of signal transduction pathways and are essential in cell-environment communication and cell response to stimuli[2,3,4]
We show the relative arrangement of major intraflagellar transport (IFT) particle subcomplexes: IFT-A, IFT-B and Bardet–Biedl Syndrome (BBS), and localization of the IFT motor protein, kinesin II, as well as tubulin cargo binding sites
We used stochastic optical reconstruction microscopy (STORM) microscopy to reveal the localization of selected Tetrahymena transition zone (TZ) proteins and to compare their spatial organization within the TZ region with the arrangement of TZ proteins described in mammalian motile and primary cilia
Summary
Cilia are microtubule-based, finger-like cell protrusions which can be roughly divided into two categories: the motile cilia whose primary function is cell motility or translocation of materials along the surface of the ciliated epithelial cell[1] and the sensory cilia which house a variety of signal transduction pathways and are essential in cell-environment communication and cell response to stimuli[2,3,4]. Recent emergence of several super-resolution imaging tools that are suitable to pinpoint subtle differences in the arrangement and distribution of proteins enabled the detailed analyses of some of the TZ, IFT and TF proteins[31,32,33,34,35] It is still unclear where exactly the IFT and BBS proteins assemble to form particles and dock before they gain access to the cilium. We studied the spatial organization of TZ, IFT and BBS proteins at the cilia base of a ciliate Tetrahymena thermophila This unicellular eukaryote assembles several hundred cilia[36,37] which can be visualized in lateral (side) and top views. Such a high number of identical structures within a single cell facilitates averaging of many super-resolution images to increase localization accuracy and enables multi-dimensional reconstruction
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