Project Summary/Abstract Autosomal dominant polycystic kidney disease (ADPKD) is predominately caused by mutations in PKD1 and PKD2. These two transmembrane proteins likely form a receptor-channel complex. Malfunction of the PKD1/2 complex in the membrane of primary cilia of kidney epithelial cells is widely thought to be the cause of the disease. However, the nature of the in vivo stimulus sensed by the PKD1/2 complex, the mechanism of channel opening and the down-stream signaling events remain uncertain. PKD2 is a transient receptor potential (TRP) channel that is also present in the ciliary membrane of many protists indicating that it has a conserved role in cilia. We propose to use Chlamydomonas as a simple system to analyze the assembly and function of the PKD2 channel in motile cilia. Our preliminary data show that Chlamydomonas PKD2 targets and anchors mastigonemes, hair-like glycoprotein polymers, to the extracellular surface of cilia. Mastigonemes are missing from a novel pkd2 null mutant, which swims with reduced velocity indicating a motility related function of PKD2 in motile cilia. Remarkably, PKD2 is anchored on just two of the nine doublet microtubules (DMTs), i.e., DMTs 4 and 8, which positions the two rows of PKD2-mastigoneme complexes perpendicular to the plane of the ciliary beating. Association to the cytoskeleton and extracellular components are typical for many mechanically gated channels. Thus, our findings suggest a mechanosensory role of PKD2 in motile cilia. In Aim 1, we propose to identify the composition of the linker that connects PKD2 to the ciliary microtubules in Chlamydomonas. We will analyze the composition of PKD2 complexes isolated from cilia, identify proteins in the vicinity of PKD2 using in vivo proximity labeling, and determine the intracellular parts of PKD2 that contribute to microtubule anchoring. The results will establish how PKD2 is targeted to just two of the nine axonemal doublets. We expect insights into how cells establish and identify differences between the DMTs, a likely requirement for complex ciliary beat patterns in general. In Aim 2, we will analyze how ciliary motility is affected in the pkd2 mutant using high speed video. Cytoskeletal and extracellular anchors can function as gating springs that open channels upon deformation. The isolation of mutants defective in PKD2 tethering will allow us to determine the role of PKD2 anchoring and patterning for ciliary motility. Calcium imaging of adhered cells during mechanical stimulation of cilia will be used to gather direct insights into PKD2’s channel function. Finally, we will determine how the distribution of PKD2 adapts to changes in the cell’s environment. Overall, we will test the hypothesis that PKD2 senses the active bending of motile cilia and that regular PKD2 arrays are required for this process. Understanding PKD2 function in motile cilia could aid in determining the mechanism of PKD2 function in primary cilia since sensing of flow-...