Abstract
BackgroundThe 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 (Ca2+) increases in kidney epithelia depend on primary cilia and primary cilium-localized Ca2+-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 Ca2+ 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 Ca2+-permeable channels that may mediate flow-induced Ca2+ entry: PC2, TRPV4, and PIEZO1.ResultsHere, we report the first measurements of Ca2+ signaling within osteocyte primary cilia using a FRET-based biosensor fused to ARL13B. We show that fluid flow induces Ca2+ increases in osteocyte primary cilia which depend on both intracellular Ca2+ release and extracellular Ca2+ entry. Using siRNA-mediated knockdowns, we demonstrate that TRPV4, but not PC2 or PIEZO1, mediates flow-induced ciliary Ca2+ increases and loading-induced Cox-2 mRNA increases, an osteogenic response.ConclusionsIn this study, we show that the primary cilium forms a Ca2+ microdomain dependent on Ca2+ 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13630-015-0016-y) contains supplementary material, which is available to authorized users.
Highlights
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
Y4 osteocyte-like cells are independent of primary cilia and stretch-activated channels, which is different from kidney cells [14]. While these results suggest that the osteocyte primary cilium-regulated mechanism of mechanotransduction is not linked to intracellular Ca2+ levels, it is unknown if the local primary cilium Ca2+ environment is distinct from the cytosol
Arl13b-linker-Ca2+ biosensor detects ciliary Ca2+ For this study, we developed a novel primary ciliumlocalized, fully ratiometric biosensor using the modified YC3.6 Ca2+-sensitive fluorescence resonance energy transfer (FRET)-based biosensor (CaB) containing ECFP and YPet fused to ARL13B
Summary
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. We directed a fluorescence resonance energy transfer (FRET)-based Ca2+ 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 Ca2+-permeable channels that may mediate flow-induced Ca2+ entry: PC2, TRPV4, and PIEZO1. The dependence of flow-induced Ca2+ increases on kidney epithelia primary cilia and the presence of mechanosensitive Ca2+-permeable channels on the ciliary membrane suggest that mechanical loading opens stretch-activated ion channels on the primary cilium that mediate Ca2+ entry. Blocking ryanodine receptors inhibited cytosolic Ca2+ increases without affecting the flow-induced ciliary Ca2+ response [18,19] These recent flow studies on kidney epithelia primary cilia demonstrate that fluid flow activates PC2 through which extracellular Ca2+ enters and triggers ryanodine receptors in Ca2+-induced Ca2+
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