The primary cilium orchestrates chondrocyte mechanotransduction

Purpose: to understand the role of the chondrocyte primary cilium in mechanotransduction. The primary cilium is a singular organelle protruding outward from the mature centriole into the extracellular space, decorated with many receptors and integrins. The tubulin scaffold, ciliary membrane and proteome are constructed and maintained by dynein motors and intraflagellar transport cargos. In many cell types such as kidney epithelia, vascular endothelium and osteocytes, primary cilia are involved in mechanotransduction, often through the activities of polycystin 1 and 2 located on the cilium. Physiologically cartilage experiences mechanical signals, altering cell behaviour, that include compression and fluid flow. It is thought aberrant mechanotransduction may be one factor in the development of tissue pathology. Here we test the hypothesis that the cilium is essential for chondrocyte mechanotransduction and the up-regulation of proteoglycan synthesis through an established purinergic pathway involving the release of ATP and subsequent activation of Ca2+ signalling.Methods: to test this we used a hypomorphic mutation of Tg737, which encodes for IFT88, and abolishes genesis and growth of the cilium and which has been shown in vivo to result in murine matrix patterning defects. A well-established 3D agarose culture system was implemented to allow compressive loading of murine WT and Tg737 chondrocytes in culture followed by the quantification of ATP release with a luciferase assay, calcium transients by means of Fluo-4 imaging, and matrix production by qPCR and biochemical assay. Additionally expression of purinergic receptors (P2R) and polycystins (PC) 1 and 2 were assessed by western blot and immunocytochemistry.Results: compression of WT chondrocytes increased calcium transients (p<0.05) and matrix production at gene and protein levels (p<0.05) however these mechanosensitive responses were not present in Tg737 chondrocytes. Mechanosensitive ATP release (p<0.01) was maintained between WT and Tg737 cells implying that the cilium is required for ATP reception or transduction. Indeed, exogenous addition of ATP up-regulated Ca2+ transients in WT (p<0.001) but did not in Tg737 cells, although there were no differences in P2R expression. In Tg737 cells PC-1 expression was altered such that the full size protein product was absent.Conclusions: we conclude that the primary cilium is essential for chondrocyte mechanotransduction through the regulation of purinergic Ca2+ signalling. We speculate that this may be attributed to a role for the cilia protein polycystin-1. This demonstrates the central role for the chondrocyte primary cilium in cartilage physiology in the context of the chondrocyte response to mechanical stimuli

Kinesin-II Is Required for Flagellar Sensory Transduction during Fertilization in Chlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt−) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt− flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32°C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32°C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32°C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32°C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles