In:
Science Signaling, American Association for the Advancement of Science (AAAS), Vol. 6, No. 306 ( 2013-12-17)
Abstract:
The primary cilium is a microtubule-rich, nonmotile organelle that protrudes from epithelial cells. Abnormal ciliogenesis produces pleiotropic developmental phenotypes and contributes to a class of diseases known as ciliopathies. Receptors for the secreted cell signaling ligands Wnt and Shh (sonic hedgehog) localize on ciliary membranes, and ciliary dysfunction disrupts these pathways. A pair of studies from the Clapham laboratory found that polycystin proteins, encoded by polycystic kidney disease (PKD)–associated genes, heteromultimerized to form calcium-permeable channels on cilia and that changes in ciliary calcium, which occurred independently of cytosolic calcium, modulated Shh pathway activity. Using whole-cell patch-clamp analysis of cilia in four different human and mouse epithelial cell types expressing ciliary-targeted green fluorescent protein (GFP) fusion proteins, DeCaen et al . identified an outward-rectifying, noninactivating current in cilia that were either attached to or detached from the cell body. Ciliary current density was more than 50 times as high as that measured on the cell body, and the calculated channel density was comparable to that of other excitable plasma membranes. In perforated patch recordings, ciliary currents were blocked by the nonselective channel antagonists gadolinium or ruthenium red and were activated by extracellular uridine triphosphate or adenosine di- or triphosphate or by cell-permeable inhibitors of calmodulin, indicating that these channels are likely activated by purinergic G protein–coupled receptors (GPCRs). Although the ciliary current was relatively nonselective for cations, calcium was more permeable than sodium or potassium. Ciliary current was reduced in human retina pigmented epithelial (hRPE) cells transfected with small-interfering RNAs targeting PKD1L1 or PKD2L1 and in embryonic fibroblasts from Pkd2l1 –/– mice. When coexpressed in human embryonic kidney cells, PKD1L1 and PKD2L1 coimmunoprecipitated and produced currents with biophysical and pharmacological properties similar to those detected in cilia. Because calcium-sensitive dyes, such as Fluo-4, do not penetrate into primary cilia, Delling et al. created stable hRPE cells expressing a cilia-targeted ratiometric calcium sensor (SMO-mCherry-GcAMP3) composed of a fusion of the Shh receptor Smoothened (SMO), the red fluorescent protein mCherry, and the calcium-sensitive fluorophore GcAMP3. In SMO-mCherry-GcAMP3 hRPE cells loaded with Fluo-4 in the cytosol, rupturing the tip of the cilia with a laser pulse produced a transient increase in ciliary calcium that propagated through the base of the cilia but did not increase Fluo-4 fluorescence and thus did not appreciably change total cytosolic calcium. Selectively uncaging a calcium chelator near the base of the cilium in cells with these calcium sensors and loaded with high calcium revealed that the rates of calcium diffusion within the cytosol and from the cytosol into the cilium were similar, indicating that the basal body of the cilia does not impede calcium flux. Cilia showed a higher membrane potential and higher resting calcium concentration than the cell body. These results, combined with the fact that cilia have a much smaller volume than the cell body, suggest that the cilium forms an isolated calcium signaling compartment: The flow of calcium from the cilium to the cytosol minimally affects cytosolic calcium concentration, and cytosolic calcium does not flow into the cilium at resting calcium concentrations. Pkd2l1 –/– mice had abnormal intestinal rotation characteristic of ciliary dysfunction and defects in Shh signaling. Embryonic fibroblasts of wild-type and Pkd2l1 –/– mice had comparable abundance of SMO. However, localization of the transcription factor GLI2 in the ciliary tip and the increased abundance of the Shh target GLI1 induced by the SMO agonist SAG were impaired in Pkd2l1 –/– fibroblasts. Treatment with SAG did not immediately affect ciliary current, but after 24 hours it increased the concentration of ciliary calcium and current amplitude, suggesting a compensatory effect. Thus, cilia have membranes with a high density of calcium-permeable channels, which can be further enhanced by prolonged Shh signaling, and the cilia function as calcium signaling organelles that are isolated from the cytosolic calcium signaling events. Disruption of the calcium signaling capacity of cilia impairs Shh signaling and may affect other signal transduction pathways. P. G. DeCaen, M. Delling, T. N. Vien, D. E. Clapham, Direct recording and molecular identification of the calcium channel of primary cilia. Nature 504 , 315–318 (2013). [Online Journal] M. Delling, P. G. DeCaen, J. F. Doerner, S. Febvay, D. E. Clapham, Primary cilia are specialized calcium signalling organelles. Nature 504 , 311–314 (2013). [Online Journal]
Type of Medium:
Online Resource
ISSN:
1945-0877
,
1937-9145
DOI:
10.1126/scisignal.2005006
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2013
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