Person:

Xu, Yangqing

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.

Oxysterols bind the seven-spanner transmembrane protein Smoothened and potently activate vertebrate Hedgehog signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, if oxysterols are important for normal vertebrate Hedgehog signaling, and whether antagonizing oxysterols can inhibit the Hedgehog pathway. We developed azasterols that block Hedgehog signaling by binding the oxysterol-binding site of Smoothened. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smoothened, and is completely separable from the site bound by other small molecule modulators, located within the heptahelical bundle of Smoothened. Smoothened mutants in which oxysterol binding is abolished no longer respond to oxysterols, and cannot be maximally activated by the Hedgehog ligand. Our results show that oxysterol binding to vertebrate Smoothened is required for normal Hedgehog signaling, and that targeting the oxysterol binding site is an effective strategy to inhibit Smoothened.