Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• The Hedgehog signaling pathway is crucial for cancer research and regenerative medicine.
• Key pathway proteins, including Sonic Hedgehog (SHH) and Patched1 (PTCH1), dynamically localize in primary cilia during signal transduction.
• SHH binding to PTCH1 in cilia inactivates PTCH1 and promotes Smoothened (SMO) accumulation.

Purpose of the Study:

• To investigate the single-molecule dynamics of PTCH1 in live cells during Hedgehog pathway activation.
• To understand how SHH binding affects PTCH1's movement and confinement within primary cilia.
• To explore the role of the cellular membrane microenvironment in PTCH1 regulation.

Main Methods:

• Utilized advanced single-molecule microscopy techniques to observe live cells.
• Tracked the diffusion and confinement of individual PTCH1 molecules within primary cilia.
• Compared PTCH1 dynamics after SHH treatment versus cholesterol depletion.

Main Results:

• PTCH1 molecules in cilia exhibit diffusion, directional transport, and confinement.
• SHH treatment increased PTCH1 confinement and reduced free diffusion.
• SHH treatment, unlike cholesterol depletion, enhanced the diffusion coefficient of mobile PTCH1 molecules.
• These changes suggest SHH modifies the PTCH1-associated membrane microenvironment.

Conclusions:

• SHH binding significantly alters PTCH1 motional dynamics within primary cilia.
• PTCH1 inactivation by SHH involves changes in its diffusion and confinement behavior.
• The findings point towards a modulation of the primary cilium membrane microenvironment by SHH signaling.