Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xviii, 179 pages) : illustrations.

Dissertation/Thesis Chair

Haixin Sui

Committee Members

Susan P Gilbert, Janice D Pata, Paul S Masters, Rajendra K Agrawal

Keywords

3D structure, correlative light and electron microscopy, electron tomography, epithelial cells, intraflagellar transport, primary cilium, Cilia and ciliary motion, Flagella (Microbiology), Epithelial cells, Electron microscopy, Cells

Subject Categories

Biology | Biophysics | Cell Biology

Abstract

Primary cilia are hair-like protrusions that stem from the basal bodies in the cytoplasm and extend into the extracellular space to sense signals. Intraflagellar transport (IFT) functions to transport cargo molecules into and out of the ciliary compartment to assemble, maintain, and disassemble the cilia. Accurate knowledge of the three-dimensional (3D) structure of primary cilia and precise details of the IFT profile is the foundation for understanding the sensory functions of primary cilia. This work covers three aspects of primary cilia. Firstly, we obtained and analyzed the overall 3D architecture of the complete primary cilia axoneme region using serial section electron tomography (SSET), and built a 3D model based on the density map. The results displayed that the microtubule arrangement patterns vary along the axoneme from the base to the tip in primary cilia, due to the two non-adjacent microtubule complexes (MtCs) shifting inside and the terminating of the microtubules progressively. Thus, the major part of the axoneme in primary cilia is not the well-accepted 9+0 structure. We examined the structural characteristics that are associated with the elastic bending property of primary cilia as mechanosensors, including the diameter profile of the primary cilia and the network property. The transition regions of the microtubule doublet (MtD) to singlet were investigated, and the results suggest that the inner junction of the B-tubule of the doublet plays the main role in the doublet to singlet transition. Vesicles in the ciliary membrane are observed that will be released as ectosomes for special functions. The 3D architecture of the primary cilia axoneme region provides the baseline to better understand the functional mechanisms of the primary cilia. Secondly, we obtained the 3D structure of the basal body of primary cilia in kidney cells using SSET of samples prepared with high-pressure freezing (HPF)-freeze substitution (FS) method. Our data suggest that the transition fiber is composed of fibrous structures, and it contains two branches, an inner branch and an outer branch, mainly connecting the ciliary membrane and cell membrane, respectively, at the boundary region. Several structural features were identified, including the multiple connecting filaments linking the adjacent microtubule triplets (MtTs) at the proximal region, the highly condensed luminal amorphous clusters, and some types of the luminal adjacent-MtC linkers, which serve to stabilize the structural organizations of the basal body in primary cilia. Septin-like filaments were observed, which may serve as membrane diffusion barriers for macromolecular particles. In addition, the transition regions of MtT to MtD were investigated, and nearly all of them start at the inner junction of the C-tubule in the triplet. The triplet-to-doublet transition completes before the microtubule complex extends out of the cell. Thirdly, the IFT profile in primary cilia were investigated using a correlative light and electron microscopy (CLEM) strategy, in order to answer the question of how the IFT trains move along the microtubules with the varying arrangement patterns from the base to the tip in primary cilia. Three types of IFT trains, the anterograde IFT train, the retrograde IFT train, and the standing IFT train, were determined in primary cilia. Both anterograde and retrograde IFT trains are able to move along both A- and B-tubules across multiple MtCs. The inside-shift MtCs at the region close to the ciliary membrane are still able to serve as tracks for IFT trains. These results provide insight into the IFT dynamic behavior in primary cilia.

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