Phosphoinositide lipids in primary cilia biology

Abstract

Primary cilia are solitary signalling organelles projecting from the surface of most cell types. Although the ciliary membrane is continuous with the plasma membrane it exhibits a unique phospholipid composition, a feature essential for normal cilia formation and function. Recent studies have illustrated that distinct phosphoinositide lipid species localise to specific cilia subdomains, and have begun to build a 'phosphoinositide map' of the cilium. The abundance and localisation of phosphoinositides are tightly regulated by the opposing actions of lipid kinases and lipid phosphatases that have also been recently discovered at cilia. The critical role of phosphoinositides in cilia biology is highlighted by the devastating consequences of genetic defects in cilia-associated phosphoinositide regulatory enzymes leading to ciliopathy phenotypes in humans and experimental mouse and zebrafish models. Here we provide a general introduction to primary cilia and the roles phosphoinositides play in cilia biology. In addition to increasing our understanding of fundamental cilia biology, this rapidly expanding field may inform novel approaches to treat ciliopathy syndromes caused by deregulated phosphoinositide metabolism.

Keywords: ciliopathy; phosphoinositides; primary cilia.

Figure 1.. Localisation of phosphoinositides defines membrane identity.

(A) Structure of PtdIns, arrows highlight the 3-, 4- and 5-positions of the inositol ring which can be phosphorylated. (B) Distinct phosphoinositides localise to the cytoplasmic leaflet of specific membrane domains and thereby confer membrane identity and control trafficking and signalling events. The major phosphoinositides at the membrane domains are depicted. PtdIns(4,5)P₂ is the major species at the plasma membrane, although lower levels of PtdIns(4)P are also detected and PIP₃ and PtdIns(3,4)P₂ are transiently produced following growth factor stimulation. The endocytic membranes are predominantly decorated by PtdIns(3)P which is converted to PtdIns(3,5)P₂ as vesicles progress through the endocytic network to the multivesicular body and lysosome. PtdIns(4)P is the major Golgi-resident phosphoinositide.

Figure 2.. Primary cilia structure.

The primary cilium is built around a scaffold of microtubules known as the axoneme. The microtubules that make up the axoneme are arranged as nine doublets (each doublet contains an A tubule and a B tubule). At the base, the axoneme is anchored by the basal body. The basal body is a modified mother centriole which has acquired distal appendage proteins that it uses to dock with the plasma membrane. It consists of nine microtubule triplets (A–C tubules). The axoneme is covered by the ciliary membrane, a membrane domain continuous with the plasma membrane but separated by a diffusion barrier. The ciliary membrane originates from Golgi-derived pre-ciliary vesicles and exhibits a unique lipid and protein composition relative to the plasma membrane. The membrane domain between the ciliary membrane and the plasma membrane is known as the ciliary pocket and is proposed to be the site where cargo from the endocytic network enter the ciliary membrane. A diffusion barrier at proximal end of the axoneme allows the cilium to maintain autonomy from the rest of the cytoplasm. The diffusion barrier is made up of three multi-protein complexes (MKS, NPHP and CEP290) at the transition zone, phosphoinositide binding Septins and the basal body distal appendages proteins (also known as transition fibres). The primary cilium does not synthesise its own protein components, therefore all proteins must be trafficked into and within the cilium. The IFT system mediates trafficking within the cilium. Kinesin microtubule motors walk along the axonemal microtubules and mediate anterograde IFT in complex with the IFT-B proteins which bind cargo molecules. Conversely, retrograde IFT is mediated by the microtubule motor cytoplasmic dynein and the IFT-A complex.

Figure 3.. Primary cilia dynamics.

Primary cilia assembly occurs when cells exit the cell cycle or in response to developmental signals. (i) Golgi-derived pre-ciliary vesicles dock with the mother centriole and fuse to become the ciliary vesicle. The mother centriole differentiates into the basal body by the acquisition of accessory structures and microtubule capping protein CP110 is removed to enable microtubule elongation. (ii) A nascent cilium is formed within the cytosol by microtubule assembly from the basal body which is covered by the double membrane ciliary vesicle. (iii) Finally the ciliary vesicle fuses with the plasma membrane to reveal the mature cilium projecting into the extracellular space. (iv) Primary cilia disassemble as cells progress through the cell cycle in response to growth factor stimulation. Disassembly occurs via microtubule destabilisation and disassembly. Cilia decapitation also contributes to disassembly by removing positive regulators of cilia maintenance. (v) Once the cilium has disassembled the mother centriole is released for mitotic spindle formation.

Figure 4.. Hedgehog signalling is initiated at primary cilia.

(A) In the absence of Hedgehog ligand, (i) its receptor PTCH localises to the primary cilium and represses the pathway by (ii) sequestering the GPCR-like protein SMO out of the cilium. (iii) The GLI transcription factors are sequestered in the cytoplasm. (iv) The GPCR GPR161 localises to cilia and supports the processing of GLI to the proteolytically processed transcriptional repressor. (v) GLI repressors translocate to the nucleus to inhibit target gene expression. (B) (i) Hedgehog ligand binding to PTCH1 results in PTCH1 movement out of the cilium and (ii) de-repression of SMO, which now accumulates in the ciliary membrane. SMO may also be directly activated by the small molecule ligand SAG. (iii) These events result in exit of GPR161 from the cilia and (iv) GLI accumulation at the cilia tip where it becomes activated. (v) Active GLI then translocates to the nucleus to promote Hedgehog target gene expression.

Figure 5.. Phosphoinositide map at primary cilia.

The phosphoinositides localise to discrete subcompartments of the ciliary membrane. PtdIns(4)P is the major phosphoinositide in the ciliary membrane. The transition zone membrane at the cilia base is a ‘hot-spot’ enriched for multiple phosphoinositides including PtdIns(4,5)P₂, PIP₃ and PtdIns(3,4)P₂. PtdIns(3)P is associated with the pericentriolar recycling endocytic compartment. The phosphoinositide regulatory enzymes PI3K-C2α, PIPKIγ, INPP5E, OCRL and INPP5B also localise to specific ciliary subdomains in proximity to their substrates and products.

Figure 6.. INPP5E regulates Hedgehog signalling via ciliary phosphoinositide metabolism.

(A) INPP5E localises to the cilia axoneme and transition zone. In the axoneme INPP5E prevents the accumulation of PtdIns(4,5)P₂ via hydrolysis to PtdIns(4)P. At the transition zone INPP5E regulates the local PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ levels. Upon Hedgehog stimulation of wild type cells SMO accumulates at the cilium promoting ciliary exit of the negative regulator GPR161 and subsequently Hedgehog target gene expression. (B) In the axoneme of Inpp5e knockout cilia PtdIns(4,5)P₂ accumulates. TULP3 binds the increased ciliary PtdIns(4,5)P₂ inducing GPR161 accumulation even in the presence of Hedgehog ligand stimulation. PtdIns(4,5)P₂ and PIP₃ accumulate in the Inpp5e-null transition zone upon Hedgehog pathway activation which is associated with disruption of the transition zone complexes and compromised diffusion barrier retention function. Therefore SMO fails to concentrate in Inpp5e-null cilia. These events contribute to a reduced transcriptional response to Hedgehog stimulation.

Figure 7.. Regulation of cilia stability by transition zone PIP₃.

PIP₃ localises to the transition zone and its levels increase following stimulation by growth factors possibly via an unknown class I PI3K. Increased transition zone PIP₃ activates a localised AKT signalling axis which inactivates GSK3β contributing to reduced cilia stability. Local PIP₃ is negatively regulated by the cilia-localised PI 5-phosphatases INPP5E and OCRL.