Hedgehog signals are the key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause developmental diseases, congenital heart disease, and cancers such as basal cell carcinoma. There is an urgent need to understand the molecular mechanisms that underlie the modulation of Hedgehog signaling pathway for its potential preventative and therapeutic value. It is known ver- tebrate Hedgehog signaling relies on the ciliary trafficking of Hedgehog signaling receptors, among which smoothened (SMO) is the central positive mediator of Hedgehog signaling. If mutations occur in either intrafla- gellar transporters (IFTs), or in the ciliary transition zone, SMO activities can be severely disrupted. However, the molecular mechanism of how IFT particles and transition zone regulate trafficking of SMO is currently un- known. Determination of the molecular regulation mechanisms is the objective of this application. Our prelimi- nary data acquired by Stochastic Optical Reconstruction Microscopy (STORM) showed the colocalization of transition zone proteins with SMO and IFT88, suggesting that transition zone proteins and IFT particles interact with SMO. Based on previous studies and our own primary data, my central hypothesis is that the transition zone serves as a checkpoint for Hedgehog signaling receptors, and IFTs help Hedgehog signaling receptors cross the transition zone. This transition zone checkpoint model represents a novel mechanism for the control of cilium trafficking and the cross-interaction between different ciliary cargos. It could potentially allow new ap- proaches to manipulate Hedgehog signals, and underlie the foundation for treatments of diseases caused by defects in Hedgehog signaling. To approach to the project, I plan to map SMO molecules and IFT particles in the transition zone using multicolor 3D STORM. It will reveal the spatial relationship between SMO molecules and these ciliary components at a resolution of ~15 nm. Algorithms will be developed to reduce the uncertainty of the spatial easements caused by structural heterogeneity and immunostaining, providing a ~ 5nm precision of the distance between investigated proteins, indicating protein-protein interaction. Equally important as the static structural study, I also plan to detect the interactions among SMO molecules, IFT particles, and transition zone proteins dynamically using single-particle tracking and photoconversion imaging. The proposed project will not only offer new insights into the molecular mechanisms of Hedgehog signaling regulation, but also ad- vance a suite of microscopy-based technologies and algorithms that can be broadly applied to the fields of cell signaling and structural biology. Furthermore, the results are expected to have broad impact, because the reg- ulatory components to be identified by this project will provide new mechanisms and new drug screen for pre- ventive and therapeutic interventions. 1
Defects in Hedgehog signaling can cause developmental diseases, congenital heart disease, and cancers such as basal cell carcinoma. Vertebrate Hedgehog signaling relies on the trafficking of Hedgehog signaling receptors in primary cilia. Determination of the molecular mechanisms of how primary cilia regulate the trafficking of Hedgehog signaling molecules will lay the foundation for advances in disease treatment and prevention.