Identification of ERICH3 as a novel regulator of primary cilium formation and the localisations of ciliary transport and Sonic Hedgehog signaling proteins

Abstract

Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. By employing the human retinal epithelial cell line (hTERT-RPE1), this study sought to identify new regulators of cilium formation and function, including IFT-associated proteins. From a list of previously published candidate cilia-related genes, this PhD research investigated the possible ciliary associations of an uncharacterised glutamate rich protein, ERICH3. By employing a GFP-ERICH3 construct, it was found that ERICH3 localises to the primary cilium of both transiently transfected and stable hTERT-RPE1 cells. Specifically, GFP-ERICH3 localises along with the entire axonemal length but is excluded from the basal body region. To examine possible cilium-associated roles, two independent siRNAs targeting ERICH3 were employed to deplete its levels in RPE1 cells. Using assays of ciliogenesis and cilium length, it was found that ERICH3 depletion leads to a modest reduction (25-35%) in the ability of hTERT-RPE1 cells to form a cilium, which is abnormally short. Rescue of the cilium formation and length phenotypes by an siRNA resistant ERICH3 construct confirmed the role of ERICH3 in cilium structure regulation. Consistent with the observed ciliary defects, the endogenous ciliary localisations and distributions of IFT proteins, IFT88 (IFT-B protein) and IFT140 (IFT-A proteins), were disrupted in ERICH3-depleted cells. Specifically, both proteins were found to accumulate at the ciliary tip of ERICH3-depleted hTERT-RPE1 cells, consistent with a defect in retrograde IFT. In further support of a role in retrograde IFT, reduced endogenous ciliary levels of retrograde IFT regulators, ARL13B and a BBSome component (BBS5), were observed in ERICH3-disrupted cells. In addition, using siRNAs to deplete ARL13B and the BBSome, this research revealed that ERICH3, ARL13B and the BBSome are interdependent for their localisation of hTERT-RPE1 primary cilia. Finally, the research in this thesis investigated a possible role for ERICH3 in the cilium-based sonic hedgehog signaling pathway. It was found that SMO and GPR161 abnormally accumulate in ERICH3-depleted cilia upon Shh pathway activation via SAG treatment, consistent with a defect in removing these positive and negative pathway regulators from the cilium.

Together, the work in this thesis identifies ERICH3 as a novel component of the human primary cilium. From the data, it is proposed that ERICH3 functions together with ARL13B and the BBSome within retrograde IFT-associated pathways to remove signaling proteins from the cilium. The research therefore uncovers a new player in retrograde IFT regulation and signaling.