Modulation of Ciliary Phosphoinositide Content Regulates Trafficking and Sonic Hedgehog Signaling Output

Abstract

Ciliary transport is required for ciliogenesis, signal transduction, and trafficking of receptors to the primary cilium. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) have been associated with ciliary dysfunction; however, its role in regulating ciliary phosphoinositides is unknown. Here we report that in neural stem cells, phosphatidylinositol 4-phosphate (PI4P) is found in high levels in cilia whereas phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is not detectable. Upon INPP5E inactivation, PI(4,5)P2 accumulates at the ciliary tip whereas PI4P is depleted. This is accompanied by recruitment of the PI(4,5)P2-interacting protein TULP3 to the ciliary membrane, along with Gpr161. This results in an increased production of cAMP and a repression of the Shh transcription gene Gli1. Our results reveal the link between ciliary regulation of phosphoinositides by INPP5E and Shh regulation via ciliary trafficking of TULP3/Gpr161 and also provide mechanistic insight into ciliary alterations found in Joubert and MORM syndromes resulting from INPP5E mutations.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

“…2 G and H); however, the basally high cAMP level in cilia depends on adenylyl cyclase 5/6 activity that requires PIP 3 but not stimulatory G proteins. Others have reported that PI(4,5)P 2 and INPP5E play critical roles in ciliary signaling (31) and that inhibition of PI3K prevents IGF-I-stimulated Akt and SHH-mediated increase in Gli activity in a luciferase assay (32). Additionally, cross-talk between inhibition of HH and PI3K expression was also reported in some medulloblastomas (33).…”

Role of Ocrl1 and Inpp5E in primary cilia assembly and maintenance: a phosphatidylinositol phosphatase relay system?

“…Indeed, two recent studies 8,9 specifically addressed PIP composition within the PC. While one study used PI-specific …”

“…28 The lack of PI(4,5)P 2 regulation inside the cilia is expected to result in aberrant signaling, in part due to abnormal GPCRs levels. 8 The concentration of PI(4,5)P 2 is controlled by both 5′ kinases and phosphatases. 7 However, besides immunofluorescence-based detection of PIP5Kγ at the ciliary base, 8 no information is available concerning the localization and function of PIP5 kinases in the PC.…”

“…Recent studies have also unveiled the PIP content of the specialized signaling organelle known as the primary cilium, 8,9 which has a strikingly different PIP content from the PM, though in principle they are continuous with each other. The following section will highlight the importance of PIP regulation in the maintenance of the PC.…”

“…Madhivanan et al antibodies to examine the neural stem cell cilium, 8 the other relied on genetically encoded markers known to bind PI4P (EGFP-2xp4M SidM ) and PI(4,5)P 2 (EYFP-PH PLCδ1 ) in different cell lines. 9 Both these studies concluded that the major PIP in the cilium is PI4P, while PI(4,5)P 2 is restricted to the ciliary base.…”