The primary cilium functions as a mechanical and calcium signaling nexus

Kristen L Lee, Marie D Guevarra, An M Nguyen, Mardonn C Chua, Yingxiao Wang, Christopher R Jacobs

The primary cilium is an antenna-like, nonmotile structure that extends from the surface of most mammalian cell types and is critical for chemosensing and mechanosensing in a variety of tissues including cartilage, bone, and kidney. Flow-induced intracellular calcium ion (Ca(2+)) increases in kidney epithelia depend on primary cilia and primary cilium-localized Ca(2+)-permeable channels polycystin-2 (PC2) and transient receptor potential vanilloid 4 (TRPV4). While primary cilia have been implicated in osteocyte mechanotransduction, the molecular mechanism that mediates this process is not fully understood. We directed a fluorescence resonance energy transfer (FRET)-based Ca(2+) biosensor to the cilium by fusing the biosensor sequence to the sequence of the primary cilium-specific protein Arl13b. Using this tool, we investigated the role of several Ca(2+)-permeable channels that may mediate flow-induced Ca(2+) entry: PC2, TRPV4, and PIEZO1. Here, we report the first measurements of Ca(2+) signaling within osteocyte primary cilia using a FRET-based biosensor fused to ARL13B. We show that fluid flow induces Ca(2+) increases in osteocyte primary cilia which depend on both intracellular Ca(2+) release and extracellular Ca(2+) entry. Using siRNA-mediated knockdowns, we demonstrate that TRPV4, but not PC2 or PIEZO1, mediates flow-induced ciliary Ca(2+) increases and loading-induced Cox-2 mRNA increases, an osteogenic response. In this study, we show that the primary cilium forms a Ca(2+) microdomain dependent on Ca(2+) entry through TRPV4. These results demonstrate that the mechanism of mechanotransduction mediated by primary cilia varies in different tissue contexts. Additionally, we anticipate that this work is a starting point for more studies investigating the role of TRPV4 in mechanotransduction.