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.

Polystyrene-b-poly(4-vinylpyridine) copolymers were quaternized with 1-bromohexane and 6-perfluorooctyl-1-bromohexane. Surfaces prepared from these polymers were characterized by contact angle measurements, near-edge X-ray absorption fine structure spectroscopy and X-ray photoelectron spectroscopy. The fluorinated pyridinium surfaces showed enhanced antibacterial activity compared to their nonfluorinated counterparts. Even a polymer with a relatively low molecular weight pyridinium block showed high antimicrobial activity. The bactericidal effect was found to be related to the molecular composition and organization in the top 2-3 nm of the surface and increased with increasing hydrophilicity and pyridinium concentration of the surface.

Mice may be useful for studies of skeletal aging, but there are limited data on changes in bone structure and strength over their lifespan. We obtained bones from female and male BALB/c mice at ages 2, 4, 7, 12 and 20 months and evaluated their structural, densitometric and mechanical properties. MicroCT of the mid-diaphysis of the femur and radius indicated that during skeletal growth (2-7 months) bone cross-sectional size (area, moment of inertia) increased rapidly; during aging (7-20 months) cortical area was maintained while moment of inertia continued to increase. Bones from females were smaller than males at young ages, but not at later ages. Changes in wholebone stiffness and strength reflected the changes in bone size, with a rapid increase from 2-7 months followed by little or no change. In contrast, energy-to-fracture declined with aging. Cortical tissue mineral density (TMD) increased during growth and was maintained with aging. MicroCT of trabecular bone revealed age-related changes that were site-dependent. The proximal tibia showed a clear pattern of age-related decline in trabecular BV/TV, with progressive decreases after 4 months in both sexes; lumbar (L5) vertebra had more modest age-related declines; in contrast, caudal (Ca7) vertebra had increasing BV/TV with aging. Overall, we found no evidence that females had more pronounced age-related deterioration than males. We conclude that bones from aging female and male BALB/c mice exhibit many of the changes seen in humans, and are therefore a clinically relevant model for studies of skeletal aging.

Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction.