Elucidating the Architecture and Mechanism of the Ciliary Gate

University Of California, San Francisco, San Francisco CA

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Abstract

DESCRIPTION (provided by applicant): Cilia are evolutionarily ancient organelles that can move and receive signals from their surroundings, much like antennae. Depending on the cell type, cilia can transduce mechanosensory signals or developmental signals, or propel extracellular fluid. Cilia with different functions are distinguished by distinct ciliary proteins.A region at the base of cilia called the transition zone (TZ) is critical for the regulation of ciliay protein composition. Disruption of TZ function causes a variety of ciliopathies, including polycystic kidneys, neural tube defects, and other severe developmental disorders. Despite its central role in ciliary function, how the TZ regulates ciliary composition is unknown. To address major gaps in cilia biology I will answer four questions. 1) What is the structural basis of the ciliary gate? To address this question, I will use three-dimensional Stochastic Optical Reconstruction Microscopy (3D-STORM) and Structured Illumination (SIM) to determine TZ architecture in situ in cultured primary mammalian cells. 2) What is the molecular mechanism of ciliary gating? I will use a combination of superresolution microscopy and mutational analysis to assess how access of ciliary proteins differs upon TZ perturbation. 3) How do ciliopathy mutations compromise TZ structure? I will use cryo-electron microscopy of TZs isolated from Tetrahymena thermophila to resolve how ciliopathies alter the TZ structure. 4) Do cilia with distinct functions have TZs with unique compositions? I will analyze the subcellular localization of TZ proteins in Tetrahymena and perform functional assays of specialized cilia. I hypothesize that distinct structures within the TZ comprise distinct functional modules. I also seek to elucidate how cilia with different functions, such as motile versus sensory cilia, diversify their roles potentially by differential expression of specific TZ components. My preliminary superresolution microscopy studies have revealed the precise location of several TZ components, providing the first insights into the architecture of the TZ. In addition, I have found that the TZ proteins Tectonic 2 and B9d1 localize to distinct subsets of cilia in Tetrahymena. Further investigation of TZ proteins that potentially regulate the formation, signaling, or motilit of alternate cilia in Tetrahymena will inform how specialization of cilia is achieved. Because cili organization is critical to its cellular function, establishing the TZ architecture will reveal the structural basis of ciliary gating, as well as its overall role in cell signaling and motility. Importantly, this proposed work will shed light on the mechanisms by which defects in TZ structure cause ciliopathies, and a precise molecular map of the TZ will serve as a framework for the design of novel treatments for ciliary diseases.