Intraflagellar transport process in primary cilium maintenance

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PROJECT SUMMARY Primary cilia are non-motile antenna-like sensors that protrude into extracellular space for detecting a wide range of signals, including signals for cell proliferation control. Problems in primary cilium assembly and structure/length maintenance, and failures to distribute ciliary sensory proteins to specific locations of the cilium, are linked to a wide variety of medical disorders and developmental abnormalities (termed ciliopathies). Assembly and maintenance of the ciliary framework (axoneme) and distribution of sensory proteins in the primary cilium rely on motor-driven intraflagellar transport (IFT) trains that travel along axonemal microtubule complexes (MtCs). Knowing details about IFT processes in the primary cilium is an essential foundation for understanding the proper distribution of sensory proteins in the primary cilium. Understanding the functional roles of IFT processes in primary cilium assembly and maintenance is fundamental for studies of related ciliopathies. The objectives of this proposal are to determine how anterograde IFT trains move from the base along the axoneme to reach the cilium tip, and then transition into the retrograde trains to move back in primary cilia, and how their activities contribute to maintenance of the structure/length of the primary cilium axoneme. The long-term goal of this proposal is to develop a mechanistic understanding of the functional roles and associated physiological processes of primary cilia in tissue homeostasis and ciliopathies. Our central hypothesis is that the behavior and architecture of IFT trains in primary cilia differ from that in motile flagella. We will test our central hypothesis in three specific aims: 1) Determine how IFT trains travel along the primary cilium axoneme by a correlative study using both light microscopy (LM) and cellular electron microscopy (EM); 2) Determine how continuous IFT contributes to primary cilium maintenance by serial section electron tomography (SSET) and three-dimensional (3D) image analysis; and 3) Define the structural units and conformational changes of the IFT trains at the cilium tip by cryo- electron tomography and 3D image analysis. We will pursue an innovative strategy that combines 3D cell culturing methods and light microscopy with techniques of 3D-EM specimen preparation and data analysis to overcome experimental difficulties for studying primary cilia of epithelial cells with inhibitable IFT. The proposed research is significant because it will provide critical knowledge about the process of IFT trains moving along the primary cilia, define the roles of IFT in primary-cilium structure maintenance, and offer the first structural-level description of the molecular environment at the primary cilium tip that facilitates the IFT anterograde-retrograde transition, cargo release, ectosome budding, and other important molecular events for physiological functions of primary cilia. The impact of the proposed studies is broad, as the anticipated knowledge will become a previously unavailable reference for studies of primary cilium functional mechanisms in development, organogenesis, tissue homeostasis and wound repairing.