Time-resolved proteomics profiling of the ciliary Hedgehog response.

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The primary cilium acts as a critical signaling hub, interpreting extracellular cues like Hedgehog (Hh) signals.
• Hedgehog pathway activation involves dynamic changes in the protein, lipid, and second messenger composition of the primary cilium.

Purpose of the Study:

• To comprehensively profile the time-dependent changes in the ciliary proteome in response to Hedgehog signaling.
• To identify novel proteins involved in Hedgehog signal transduction within the primary cilium.

Main Methods:

• Utilized proximity labeling of cilia coupled with quantitative mass spectrometry.
• Applied a time-resolved proteomic approach to analyze ciliary composition changes upon Hh pathway activation.

Main Results:

• Successfully identified known Hh-regulated ciliary proteins and discovered two additional proteins.
• Revealed the rapid exit of a cyclic adenosine monophosphate-dependent protein kinase (PKA) regulatory subunit and G protein-coupled receptor GPR161 from cilia upon Hh stimulation.
• Identified the phosphatase Paladin as a novel, cell type-specific regulator that enters primary cilia during Hh pathway activation.
• Proposed a GPR161/PKA module for sensing and amplifying cyclic adenosine monophosphate (cAMP) signals.

Conclusions:

• Quantitative ciliary proteome profiling is a powerful, unbiased method for studying primary cilia signaling.
• The findings provide new insights into the molecular mechanisms of Hedgehog signaling and cAMP regulation within cilia.
• This approach holds promise for accelerating the characterization of ciliopathies and their associated signaling defects.