Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.