title: The primary cilium functions as a mechanical and calcium signaling nexus.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, MC 8904, 1210 Amsterdam Ave, New York, NY 10027 USA.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Lee
authors.affiliation: Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, MC 8904, 1210 Amsterdam Ave, New York, NY 10027 USA.
authors.forename: Marie D
authors.initials: MD
authors.lastname: Guevarra
authors.affiliation: Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, MC 8904, 1210 Amsterdam Ave, New York, NY 10027 USA ; Jacobs Technion-Cornell Innovation Institute, Cornell Tech, New York, NY 10011 USA.
authors.forename: An M
authors.initials: AM
authors.lastname: Nguyen
authors.affiliation: Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, MC 8904, 1210 Amsterdam Ave, New York, NY 10027 USA ; Department of Biotechnology, University of British Columbia, Vancouver, BC V6T 1Z4 Canada.
authors.forename: Mardonn C
authors.initials: MC
authors.lastname: Chua
authors.affiliation: Bioengineering Department, UC San Diego, La Jolla, CA 92093 USA.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, MC 8904, 1210 Amsterdam Ave, New York, NY 10027 USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Jacobs
keywords: Biosensor; Calcium signaling; Kidney epithelia; Mechanotransduction; Osteocyte; Primary cilium