Mutations in the genes encoding polycystin-1 (PC1) and polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N-terminal fragment that remains noncovalently attached to the transmembrane C-terminus. Exposing cells to alkaline solutions elutes the N-terminal fragment while the C-terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a "strip-recovery" synchronization protocol to study PC1 trafficking in polarized LLC-PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans-Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by-pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.

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Pathological mutations in PKD1 and PKD2 cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PKD1 and PKD2 code for Polycystin 1 (PC1) and Polycystin 2 (PC2), respectively. PC1 and PC2 traffic to the primary cilium in epithelial cells, including LLCPK cells. They assemble with one another to form a complex that may mediate aspects of ciliary signaling. It has been shown that, in both ciliated and non‐ciliated cell lines, expression of PC2 is necessary in order to permit PC1 to traffic to the cell surface membrane. To identify potential regulatory partners of PC1, we conducted a genome wide siRNA knockdown screening study in HEK 293T cells. This analysis suggested that knockdown of the protein phosphatase, calcineurin, decreased PC1 cell surface localization. Interestingly, in C. elegans, dephosphorylation of PC2 by calcineurin has been shown to be necessary for PC2 trafficking to the male sensory neuronal cilia. We find that inhibition of calcineurin through Cyclosporine A treatment in LLCPK cells produced a decrease of PC1 and PC2 accumulation in the primary cilium. In addition, Cyclosporine A‐treated zebrafish embryos develop a curly tail phenotype that is characteristic of zebrafish in which functional expression of PKD1 a/b or PKD2 has been disrupted. We hypothesize that calcineurin may play a role in transport of the PC1/PC2 complex to the primary cilium, perhaps by mediating the dephosorylation of PC2.