ILLUMINATING THE CELLULAR ANTENNA: PRINCIPLES OF CILIARY SIGNALING DISCOVERED BY MOLECULAR SENSORS AND ACTUATORS
Phua, Siew Cheng
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Primary cilia function as specialized sensory antennae for cells to detect signals critical to proliferation and differentiation. Aberrant ciliary signaling is associated with developmental disorders commonly known as ciliopathies, as well as tumorigenesis. Understanding the principles of ciliary signaling is fundamental to developing strategies to treat cilia-related disorders. Yet, conventional imaging methods cannot sufficiently resolve ciliary signaling events occurring within a femtoliter volume from the cell body, and genetic or chemical perturbation are frequently non-specific. In this dissertation, our goal is to engineer molecular tools to illuminate ciliary signaling mechanisms and how they regulate cellular functions. We first developed a synthetic system for rapid, chemically-inducible trapping of protein probes in cilia. This system empowered us to discover a diffusion barrier at the ciliary base which regulates flux between the cilia and cytosol. We also built a robust series of genetically-encoded cilia-targeted calcium indicators and pioneered the visualization of ciliary calcium signals upon chemical and mechanical stimulation. Additionally, we established genetically-encoded actuators to manipulate phosphoinositides and actin in cilia. Using these tools, we proceeded to deconstruct primary cilia machinery. We revealed that an absence of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) constitutes a fundamental aspect of primary cilia structural and functional identity. We first determined that PI(4,5)P2 depletion is required for ciliary transduction of Hedgehog signals. Next, we discovered that PI(4,5)P2 re-organization triggers actin polymerization in cilia, which excises cilia tips as extracellular ciliary vesicles in a process we call cilia decapitation. These conceptually new findings challenge currently-accepted models of cilia disassembly, and deliver novel concepts in organelle biogenesis. Moreover, we revealed that cilia decapitation occurs in quiescent cells, and drives G0 to G1 transit through Gli transcription factor activation. These findings propose decapitation-induced mitogenic signaling as a novel molecular link between the ciliary life cycle and cell-division cycle. Overall, we have established a niche in cilia biology field by specifically focusing on ciliary signaling visualization and manipulation. The molecular strategies used in our studies are relevant to a broad, interdisciplinary audience. Importantly, the principles of ciliary signaling we discovered establish a solid ground for understanding disorders caused by sensory defects of the cellular antennae.