Identification of signaling pathways regulating primary cilium length and flow-mediated adaptation

Abstract

The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

Figure 1. Increase of cilium length induced by modulation of intracellular cAMP and Ca²⁺ levels

(A) IMCD cells showed cilium elongation after increasing cAMP (100 μM forskolin) or inhibiting intracellular Ca²⁺ entry (30 μM Gd³⁺) for three hours. Acetylated α-tubulin was used as a ciliary marker (red). Scale bar, 5μm. (B) Histograms of cilia length distribution in untreated and treated for 3 hours IMCD cells (n > 150). (C) Average length of cilia of IMCD, MEK and BME cells after 3 hours of drug treatment (*P< 0.001). Error bars indicate mean ± s.d.

Figure 2. Pathway analysis of second messenger induced cilium length increase

(A) Cilium length increased after blockade of Ca²⁺ entry (Gd³⁺), inhibition of Ca²⁺ release from intracellular stores (Dantrolene) and activation of adenylyl cyclases (Forskolin) or PKA (8-Br-cAMP). Activation of Ca²⁺ signaling by Triptolide or Thapsigargin or inhibition of PKA signaling (Rp-cAMPS, KT-5270, H-89) resulted in cilium length reduction. 1,9-dideoxy-Forskolin (Dideoxy-F) and DMSO are control treatments that did not affect cilium length; * P< 0.001; ** P< 0.05. All treatments were for three hours. (B) Gd³⁺ or forskolin treatment resulted in increased intracellular cAMP; *P< 0.01, **P< 0.001. (C) Steady-state levels of intracellular Ca²⁺ were reduced under treatment of Gd³⁺ or forskolin. (D) Inhibition of PKA along with Gd³⁺ or forskolin treatment resulted in cilium length reduction (*P< 0.05). The increase of intracellular calcium (Thapsigargin) suppressed the effect of direct adenylate cyclase activation (forskolin) on cilium length but had no effect on the cilia lengthening caused by the direct stimulation of PKA (8-Br-cAMP). (E) Silencing of AC5 or AC6 prevents cilium lengthening in response to Gd³⁺ but not PKA activation by 8-Br-cAMP. The bar graph represents the average length of cilia after 48 hours of siRNA-mediated knockdown, followed by 3 hours of drug treatment. siRNA GAPDH was used as a control for siRNA targeting. n=100, (*P< 0.001, **P< 0.05). Error bars indicate mean ± s.d.

Figure 3. Anterograde IFT velocities increase under cilium lengthening

(A) Visualization of the IFT88-EYFP particles in live IMCD cells revealed a punctuate distribution throughout the ciliary axoneme. White arrows highlight particle movement (Cilium tip – Single arrowhead, Basal body – double arrowhead). Time given in seconds. Scale bar is 5 μm. (B) Drug treated cells show accumulation of IFT88 at the tips of primary cilia. Scale bar is 5 μm. (C) To determine the IFT particles velocities, time-lapse image sequences were assembled into kymographs. Black and white lines on the kymograph indicate anterograde and retrograde velocities, respectively. Length Scale bar (horizontal), 5μm. Time scale bar (vertical), 5 sec. (D) Histograms of the particle velocity in untreated and drug-treated cells. Negative velocities (left) represent retrograde particles, and positive velocities (right) indicate anterograde particles. Arrows represent mean velocity in untreated cells. (E) Ca²⁺ inhibition or cAMP activation increased anterograde velocity with no significant effect on retrograde velocity. Error bars indicate mean ± s.d.; *P< 0.001.

Figure 4. Cilium length and cAMP adaptation under flow depends on polycystins

(A, B) Untreated cells subjected to fluid flow (shear stress 0.75 dyne/cm²) (light grey bars – IMCD no flow, dark grey bars – MEK no flow, black bars – flow) underwent a significant decrease in cilium length. Treatment with forskolin in combination with flow also reduced cilium length. Fluid flow did not suppress length increase caused by Gd³⁺. (C, D) In untreated or forskolin stimulated cells cAMP levels were reduced under flow condition in wild-type cells. There was no change in cAMP in Gd³⁺ treated cells under flow. (E, F) Cilium length did not decrease under the application of flow to cells knocked down for PC-2 (IMCD^PC2KD) or harbouring genetic mutations in PC-1 (MEK^PC1mut) (hatched bars – no flow; black bars – flow) in either untreated or forskolin stimulated cells. In addition, IMCD^PC2KD and MEK^PC1mut cells did not increase cilium length in response to Gd³⁺. (G, H) cAMP levels were unchanged after flow in IMCD^PC2KD and MEK^PC1mut cells or in response to Gd³⁺. Error bars indicate mean ± s.d.*P<0.05; **P<0.001.