Defects in cilia formation or maintenance have been associated with degeneration of multiple organs including the eye, brain and kidney (55
). A spectrum of the ciliopathies range from mild to severe, including Usher Syndrome, Bardet–Biedl Syndrome, Joubert Syndrome, Leber congenital amaurosis and Merkel Syndrome (3
). This study explored a novel role of inositol phosphatase and phosphoinositide signaling components in the formation and function of the primary cilium. We show that the OCRL protein, whose gene mutations cause Lowe and Dent syndromes, is localized to the primary cilia and involved in ciliary function.
Previously, OCRL has been shown to localize in a number of subcellular compartments, including the TGN and endosomes (7
). Our study provides support that phosphoinositides play an important role in regulating the primary cilium. Using multiple approaches, we show that OCRL localizes to both the basal body and the transition zone of the primary cilium; the distribution of OCRL to the basal body and the transition zone of the primary cilia are consistent with several other ciliary proteins (64
). Additionally, our zebrafish studies showed that ocrl
morphants developed microphthalmia and microlentis, which has been observed in patients with Lowe syndrome (66
); we hypothesize that the mechanism of observed microphthalmia may be related to a defective AKT signaling cascade seen in INPP5E-associated ciliopathies (42
). INPP5E mutations in the 5-phosphatase domain are associated with Joubert and MORM syndromes (42
); similarly, mutations in the 5-phosphatase domain of OCRL also result in shorten cilia, suggesting that the inositol phosphatase activity is important in cilia maintenance. In addition, we showed that the loss of the RhoGAP domain in OCRL affects its ciliary localization, which may be due to its interaction with the lipid membrane of vesicles.
RAB GTPases are important regulators of vesicular trafficking and polarity formation (47
), and OCRL is known to interact with a number of RAB proteins through its RAB-binding domain (7
). Interestingly, RAB8 has been recognized as a common RAB GTPase for both OCRL and INPP5B (68
), and our study shows similar temporal recruitment of both OCRL and RAB8A to the primary cilia. Since RAB8A is a key regulator of cilia development, it is not surprising that the interaction between OCRL and RAB8 is important for its ciliary function.
The identification of OCRL in the primary cilia may provide a novel mechanism to explain the pathophysiology of Lowe syndrome, but potentially also Dent syndrome. The phenotypes seen in patients with Lowe syndrome, such as congenital cataract and glaucoma, renal dysfunction and neurological defects, all involve highly polarized cell types, such as renal or ocular epithelial cells. Subsequently, it follows that polarity dysregulation in the renal and neurological tissues, which can cause primary cilia signaling defects, could be responsible for the different phenotypes observed in Lowe syndrome (69
). We propose that the endocytic defects observed in OCRL knockdown cells may be a consequence of abnormal polarity regulation at the apical/basolateral membrane (17
). Given that OCRL has been shown to localize to the adherens and tight junctions in epithelial cells (8
), the breakdown of polarity in these cells may result in the mistrafficking of endocytic vesicles. Interestingly, INPP5B, a paralog of OCRL, was shown to be a positive regulator of cilia length in a large functional genomics screen of modulators of the primary cilia (70
). Thus, we propose that inositol phosphatases such as OCRL and INPP5B both regulate ciliary function.
While our in vitro
studies support a role of OCRL in ciliary function, the lack of phenotype in the murine knockout model can be interpreted in the context of the primary cilia and polarity dysfunction (71
). The knockout model of OCRL did not exhibit phenotypes similar to those seen in humans and the OCRL/INPP5B
double knockout resulted in embryonic lethality, leading to the hypothesis that INPP5B
may compensate for OCRL
in the mouse model (70
). Recently, Bothwell et al
) developed a murine model of Lowe syndrome that is an OCRL/INPP5B
double knockout with the human OCRL
knock-in to rescue the lethal phenotype. In light of our findings, it will be important to examine the function of ciliated cells in the murine models of inositol phosphatase with respect to processes such as eye and kidney development.
Because of OCRL knockout mouse did not exhibit a phenotype, we examined the function of ocrl
in motile cilia of zebrafish and presented data to show decreased cilia numbers in the KVs in ocrl
morphant animals. We suggest that OCRL may assist in the recruitment, modification and distribution of critical components of the cilia machinery essential for renewal of the cargo moieties, therefore playing a role in regulating the length of the primary cilium. The maintenance of cilia length can impact the subtle effects of signaling cascades on cellular physiology. OCRL may be added to a growing list of proteins such as IFT components and RPGR that regulate cilia length (33
). Future work is needed to further clarify the role of ciliary proteins in maintaining the polarized distribution of ciliary membrane proteins (76
During the preparation of this manuscript, Coon et al
) also showed the localization of OCRL to the cilia and a similar finding of shortened ciliary length in Lowe patient fibroblasts. Our findings agree with their finding; furthermore, using human tissue sections and animal tissue, we showed that OCRL can distribute to the cilia in the absence of RAB8A overexpression and this localization occurs in a number of ciliated cell types, including in ocular cells. In addition, our mutant analysis shows an important role for the 5-phosphatase domain of OCRL in its ciliary localization.
Based on our results, we postulate that the diverse phenotypes present in Lowe syndrome are due to the dysregulation of the primary cilia, with polarity defects in a number of cell types in this genetic disorder. The defects in the protein trafficking in the development and maintenance of the cilia may be the underlying cause for Lowe and Dent syndromes.