In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model.

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Autosomal dominant polycystic kidney disease (ADPKD) is primarily caused by mutations in the PKD1 gene.
• The PKD1 gene encodes Polycystin-1 (PC1), a protein with poorly understood localization and function.
• Directly visualizing PC1 and identifying its interacting partners has been a significant challenge.

Purpose of the Study:

• To create and validate a novel mouse model for studying endogenous Polycystin-1 (PC1).
• To identify PC1's in vivo binding partners using affinity-purification mass spectrometry (AP-MS).
• To investigate the subcellular localization and functional interactions of PC1.

Main Methods:

• Generated a CRISPR/Cas9-mediated knock-in mouse model with HA epitopes and eGFP fused to the endogenous Pkd1 gene.
• Utilized affinity-purification mass spectrometry (AP-MS) with nanobody-coupled beads for protein complex isolation.
• Performed network analyses on identified PC1 interactors.

Main Results:

• The engineered Pkd1 allele was confirmed to be fully functional.
• Endogenous PC1-eGFP expression was undetectable without signal amplification, necessitating advanced detection methods.
• AP-MS identified an in vivo PC1 interactome significantly enriched with mitochondrial proteins and metabolic pathway components.

Conclusions:

• The developed mouse model provides a valuable tool for ADPKD research, despite challenges in direct PC1 visualization.
• The identified PC1 interactome suggests a role for PC1 in mitochondrial function and cellular metabolism.
• Further development of sensitive detection methods and brighter tags is crucial for tracking endogenous PC1.