Rohatgi Lab In the Division of Oncology

Projects

Mechanisms of protein trafficking to primary cilia

Protein trafficking to cilia is a formidable challenge: the base of the cilium, the target zone for proteins, is over 1000-fold smaller than the plasma membrane of the cell. However, the accurate and dynamic movement of proteins into and out of cilia is crucial for ciliary signal transduction. Thus, a central challenge is to decipher the machinery that targets proteins to cilia in a signal-regulated manner.

Cilium Patched Nucleus Cilia Smo Nucleus
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Protein trafficking at cilium. The Hedgehog receptor Patched (Ptc1) is highly concentrated in the ciliary membrane of both cultured cells (top) and embryonic cells (bottom). Protein trafficking at cilium. After ligand (Shh) addition, the Hedgehog pathway protein Smoothened translocates to primary cilia. The biochemical regulation of this signal-induced change in localization is unknown.

Cilia as compartments for biochemical reactions

A remarkable feature of the Hedgehog pathway is that almost all its components, from the receptor Patched 1 to the transcription factor Gli are all localized in cilia.  However, the biochemical reactions that take place in cilia to change protein activity remain obscure.  We want to understand the how complex signaling reactions are orchestrated at this organelle.

Oxysterols can activate Hedgehog signaling. Their mechanism of action is unknown.

Biochemical Mechanisms of Hedgehog signaling

Despite its importance in development and disease, we lack a biochemical understanding of Hedgehog signal transduction.  We are probing steps in Hedgehog signaling using concentrated cell-free extracts, assays in cultured cells using novel sensors of signaling, and quantitative fluorescence microscopy.

Full activation Smo requires ciliary transport coupled to a second activation step. Solid black arrows denote the individual steps: transport to cilia (Smo1–›Smo2) is controlled by entry and exit steps denoted R1 and R2, the activation step (Smo2–›Smo3) by R3 and the exit of Smo3 from cilia by R4. The proposed site of action of various regulators of Hh signaling regulators is denoted above the kinetic scheme by light gray arrows.

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