Proximity labeling and interactomic study of primary cilia

Abstract

Cilia have recently emerged as sensory organelles present in many mammalian tissues involved in a wide variety of cellular processes such as fluid flow, cell movement as well as multiple signaling pathways. Over the last years an increasing number of human diseases have been linked to defects in cilia with autosomal‐dominant polycystic kidney disease (ADPKD) perhaps being the most prominent example. However, very little is known about the protein composition as well as the time‐resolved dynamic processes within this important organelle.To characterize the ciliary membrane‐associated proteome, we used the genetically‐engineered enzyme ascorbate peroxidase (APEX) and targeted it to the ciliary membrane (cmAPEX). With STED microscopy, we could confirm the subciliary localization of the tag specifically at the membrane of the cilium. Upon sequential addition of biotin phenol and H2O2 to the cells, APEX generates free biotin radicals that label proteins within the closest proximity, in particular at the ciliary membrane. Subsequently, cells are lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry.We identified numerous known ciliary proteins, in addition to novel ciliary membrane proteins and several actin modifying proteins. Inhibiting these actin‐modulating proteins regulated ciliary length, reaffirming the importance of actin dynamics in ciliogenesis. The results also identified ciliary proteins that are differentially regulated upon inhibition of actin polymerization and functional assays characterized their effect on ciliogenesis.cmAPEX is a versatile tool to study perturbations in this small and dynamic organelle. This approach provides quantitative analysis of the proteomic composition of the cilium and at the same time eliminates the need to isolate ciliary factions. We believe that this work will provide further insights into the cilium biology and suggest potential therapeutic targets for cilia‐associated genetic disorders.Support or Funding InformationDFG, CECAD Graduate School of Ageing.