Kurzfassung: 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]

Kurzfassung: Signal transduction often involves inputs that converge on a common protein or module of proteins. How such convergence produces divergent outputs remains a mystery. One hypothesis is that receiver proteins decode temporal input and convey this information to downstream effectors. The receptor tyrosine kinase family of receptors for ligands, such as platelet-derived growth factor (PDGF) and nerve growth factor (NGF), converge on the guanosine triphosphatase Ras to produce a range of distinct cellular outputs, including cell proliferation, differentiation, and migration. Toettcher et al. used an optogenetic approach to directly activate Ras and found that different temporal profiles of activation resulted in the phosphorylation of different sets of proteins. The optogenetic tool (opto-SOS) comprises two fusion proteins, one in which the catalytic domain of the Ras guanine nucleotide exchange factor Son of Sevenless is fused to the plant protein PIF and another in which the plant protein PhyB is tethered to the plasma membrane with a CAAX-box–type prenylation domain. When exposed to red light (in the presence of the chromophore phycocyanobilin), PIF and PhyB interact and recruit opto-SOS to the membrane, where it activates Ras. Similar to cells treated with PDGF or NGF, opto-SOS–expressing cells exposed to red light showed increased phosphorylation of endogenous ERK (extracellular signal–regulated kinase) and nuclear localization of ERK fused to a blue fluorescent protein (BFP-ERK), as well as enhanced proliferation and morphological changes. ERK activation in opto-SOS–expressing cells exhibited a graded steady-state response to different ratios of wavelengths of stimulatory and inhibitory light, and the response to light stimulation was fast (minutes), persistent, and reversible. The Ras-ERK module functioned as a high-bandwidth, low-pass filter. Light flashes with a period ranging from 4 minutes to 2 hours produced a similar gain (ratio of output to input amplitude), whereas high-frequency stimulation (periods less than 4 minutes) produced minimal output. The delay between BFP-ERK nuclear localization and light stimulation was 3 minutes, indicating an inherent phase shift in the input to output signal transmission. Antibody array–based proteomics to detect phosphorylation changes downstream of Ras-ERK activation revealed three classes of responses: (i) those that responded only to PDGF but not light, (ii) those that responded to PDGF and either transient or sustained light, and (iii) those that responded to PDGF and sustained but not transient light. The last category included phosphorylation of signal transducer and activator of transcription 3 (STAT3). Immunofluorescence analysis of cocultured parental and opto-SOS–expressing cells showed that sustained but not transient light stimulation led to the secretion of leukemia inhibitory factor that stimulated paracrine STAT3 phosphorylation. Thus, the optogenetic approach has revealed that, as in other systems such as neurotransmission, both the strength and timing of signal activation in the Ras-ERK pathway can affect the nature of the cellular response. The optogenetic approach will undoubtedly open the door to investigations into the dynamics of signaling modules that were not formerly possible. J. E. Toettcher, O. D. Weiner, W. A. Lim, Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155 , 1422–1434 (2013). [Online Journal]