The primary cilium is a self-adaptable, integrating nexus for mechanical stimuli and cellular signaling.

Mechanosensation is crucial for cells to sense and respond to mechanical signals within their local environment. While adaptation allows a sensor to be conditioned by stimuli within the environment and enables its operation in a wide range of stimuli intensities, the mechanisms behind adaptation remain controversial in even the most extensively studied mechanosensor, bacterial mechanosensitive channels. Primary cilia are ubiquitous sensory organelles. They have emerged as mechanosensors across diverse tissues, including kidney, liver and the embryonic node, and deflect with mechanical stimuli. Here, we show that both mechanical and chemical stimuli can alter cilium stiffness. We found that exposure to flow stiffens the cilium, which deflects less in response to subsequent exposures to flow. We also found that through a process involving acetylation, the cell can biochemically regulate cilium stiffness. Finally, we show that this altered stiffness directly affects the responsiveness of the cell to mechanical signals. These results demonstrate a potential mechanism through which the cell can regulate its mechanosensing apparatus.

The primary cilium functions as a mechanical and calcium signaling nexus.

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.

An experimental and computational analysis of primary cilia deflection under fluid flow.

In this study we have developed a novel model of the deflection of primary cilia experiencing fluid flow accounting for phenomena not previously considered. Specifically, we developed a large rotation formulation that accounts for rotation at the base of the cilium, the initial shape of the cilium and fluid drag at high deflection angles. We utilised this model to analyse full 3D data-sets of primary cilia deflecting under fluid flow acquired with high-speed confocal microscopy. We found a wide variety of previously unreported bending shapes and behaviours. We also analysed post-flow relaxation patterns. Results from our combined experimental and theoretical approach suggest that the average flexural rigidity of primary cilia might be higher than previously reported (Schwartz et al. 1997, Am J Physiol. 272(1 Pt 2):F132-F138). In addition our findings indicate that the mechanics of primary cilia are richly varied and mechanisms may exist to alter their mechanical behaviour.

Emerging role of primary cilia as mechanosensors in osteocytes.

The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell. This organelle has established mechanosensory roles in several contexts including kidney, liver, and the embryonic node. Mechanical load deflects the cilium, triggering biochemical responses. Defects in cilium function have been associated with numerous human diseases. Recent research has implicated the primary cilium as a mechanosensor in bone. In this review, we discuss the cilium, the growing evidence for its mechanosensory role in bone, and areas of future study.

Comparison of osmotic swelling influences on meniscal fibrocartilage and articular cartilage tissue mechanics in compression and shear.

Although the contribution of the circumferential collagen bundles to the anisotropic tensile stiffness of meniscal tissue has been well described, the implications of interactions between tissue components for other mechanical properties have not been as widely examined. This study compared the effects of the proteoglycan-associated osmotic swelling stress on meniscal fibrocartilage and articular cartilage (AC) mechanics by manipulating the osmotic environment and tissue compressive offset. Cylindrical samples were obtained from the menisci and AC of bovine stifles, equilibrated in phosphate-buffered saline solutions ranging from 0.1× to 10×, and tested in oscillatory torsional shear and unconfined compression. Biochemical analysis indicated that treatments and testing did not substantially alter tissue composition. Mechanical testing revealed tissue-specific responses to both increasing compressive offset and decreasing bath salinity. Most notably, reduced salinity dramatically increased the shear modulus of both axially and circumferentially oriented meniscal tissue explants to a much greater extent than for cartilage samples. Combined with previous studies, these findings suggest that meniscal proteoglycans have a distinct structural role, stabilizing, and stiffening the matrix surrounding the primary circumferential collagen bundles.

Noninvasive quantification of human nucleus pulposus pressure with use of T1rho-weighted magnetic resonance imaging.

BACKGROUND: Early diagnosis is a challenge in the treatment of degenerative disc disease. A noninvasive biomarker detecting functional mechanics of the disc is needed. T1rho-weighted imaging, a spin-lock magnetic resonance imaging technique, has shown promise for meeting this need in in vivo studies demonstrating the clinical feasibility of evaluating both intervertebral discs and articular cartilage. The objectives of the present study were (1) to quantitatively determine the relationship between T1rho relaxation time and measures of nucleus pulposus mechanics, and (2) to evaluate whether the quantitative relationship of T1rho relaxation time with the degenerative grade and glycosaminoglycan content extend to more severe degeneration. It was hypothesized that the isometric swelling pressure and compressive modulus would be directly correlated with the T1rho relaxation time and the apparent permeability would be inversely correlated with the T1rho relaxation time. METHODS: Eight cadaver human lumbar spines were imaged to measure T1rho relaxation times. The nucleus pulposus tissue from the L1 disc through the S1 disc was tested in confined compression to determine the swelling pressure, compressive modulus, and permeability. The glycosaminoglycan and water contents were measured in adjacent tissue. Linear regression analyses were performed to examine the correlation between the T1rho relaxation time and the other measured variables. Mechanical properties and biochemical content were evaluated for differences associated with degeneration. RESULTS: A positive linear correlation was observed between the T1rho relaxation time on the images of the nucleus pulposus and the swelling pressure (r = 0.59), glycosaminoglycan content per dry weight (r = 0.69), glycosaminoglycan per wet weight (r = 0.49), and water content (r = 0.53). No significant correlations were observed between the T1rho relaxation time and the modulus or permeability. Similarly, the T1rho relaxation time, swelling pressure, glycosaminoglycan content per dry weight, and water content were significantly altered with degeneration, whereas the modulus and permeability were not. CONCLUSIONS: T1rho-weighted magnetic resonance imaging has a strong potential as a quantitative biomarker of the mechanical function of the nucleus pulposus and of disc degeneration.