1. The role of post-translational modification of RPGR in its function and pathogenic mechanism. Mutations in the X-linked RPGR gene are a major cause of retinitis pigmentosa (RP), a blinding retinal disease due to degeneration of photoreceptors. RPGR gene codes for default and ORF15 variants, with the latter being important for photoreceptors and localizing to the connecting cilia, a structure equivalent to transition zone. RPGR-ORF15 contains a glutamic acid-rich repetitive sequence and a C terminal domain with unknown functions. Our work in the past year has shown that RPGR-ORF15 is polyglutamylated in its repetitive region. Sequence analysis of this region found multiple Glu-Gly repeats, which are known glutamylation consensus motifs in α-tubulin. RPGR-ORF15 interacts via its C terminus with a non-catalytic region of tubulin tyrosine ligase like-5 (TTLL5). Loss of TTLL5 in a mouse mutant abolishes RPGR glutamylation but leaves tubulin modifications intact. TheTtll5 mouse mutant phenocopies Rpgr null mice, developing progressive photoreceptor degeneration with early mislocalization of cone opsins. Interestingly, mutations in TTLL5 gene also cause human photoreceptor degeneration. Wild-type TTLL5, but neither related TTLs nor mutant forms of TTLL5 including those found in human patients, glutamylates RPGR in vitro. These data identify RPGR-ORF15 as a novel substrate for glutamylation and show this post-translational modification is essential for its function. Our work also uncovers the pathogenic mechanism whereby loss of RPGR-ORF15 glutamylation leads to retinal pathology in patients with TTLL5 gene mutations. A manuscript describing this work is in preparation (Xun Sun et al). 2. Role of MACF1 in retinal development. Cell polarity establishment requires a highly organized actin and microtubule infrastructure, and their coordinated actions. Microtubule actin crosslinking factor 1 (Macf1) has been shown to coordinate microtubule and actin interaction at focal adhesions, facilitating their rapid turn over in migrating cells. Here we show that Macf1 is critical for establishing apico-basal polarity and ciliogenesis. Ablation of Macf1 in the in the developing retina has no effect on neurogenesis and cell fate specifications. However, cells fail to establish correct apico-basal polarity. Photoreceptors are primarily affected, which unlike inner retinal neurons maintain a neuroepithelial configuration during development and through adulthood. In these cells, basal bodies lack docked ciliary vesicles failing to dock apically, and cilia do not emerge. A defect in ciliogenesis is also evident in other cells including cultured embryonic fibroblasts, suggesting that the ciliogenesis defect is independent of any role MACF1 may have in regulating apical junctional dynamics or integrity. Remarkably, deletion of Macf1 in adult WT photoreceptors caused reversal of basal body docking, loss of outer segments (equivalent of cilia) and photoreceptor degeneration. Our results show that Macf1 is generally required for apico-basal polarity establishment and cilia formation, and their maintenance. These findings highlight the importance actin microtubule coupling in fundamental cellular processes, mostly probably through facilitating the vectorial trafficking of vesicular compartments. (Helen May-Simera et al, manuscript in preparation). 3. Ablation of RPGR default isoform. It has been a long standing question as to whether exon 16-19 of the RPGR gene unique to the default isoform may have distinct and important functions in the eye or systemically. To answer this question directly we ablated the default form-specific exons (In collaboration with Lijin Dong, GEF). Mice lacking this isoform are viable and fertile, and display no ciliopathy. RPGR ORF15 expression in this line is higher than normal and photoreceptors show supranormal ERG amplitudes even at older ages (15 months). We have followed the mutant mice for 2 years and show with certainty that the photoreceptors of mutant mice remain healthy. Interestingly specific ablation of default RPGR causes an increase of RPGR ORF15 expression in the photoreceptors, and ectopic expression of RPGR ORF15 in brain and other non-ocular tissues. This observation suggests that that the splicing decisions for RPGR is mutually exclusive. Normally the default RPGR is expressed, and this is prevented by either removal of the splice acceptor site from the default variant (as in gene targeting) or through a change in splicing factor composition (as in photoreceptor environment). Our current hypothesis is that loss of RPGR-default does not cause a deleterious effect on photoreceptors and may even be beneficial to photoreceptors as a result of an uptick in expression of the ORF15 form. A manuscript reporting the details of this study is in preparation (Sun et al). 4. Molecular mechanisms of ciliary trafficking of proteins in photoreceptors and in primary cilia in general. This line of investigation centers at the tubby (and family of proteins) and RPGR-interaction networks. We hypothesized that loss of tubby protein may generally affect ciliary targeting of GPCR receptors including rhodopsin and cone opsin and its function is related to its PIP2 sensor function. Specifically, we postulate that that there is a sharp gradient of PIP2 levels at the preciliary membrane and the membrane enveloping the ciliary transition zone. As trafficking complexes gain entry in to the transition zone, tubby senses a sharp drop in PIP2 and falls off the membrane, triggering yet unidentified molecular cascades that complete the transport process. We further postulates that, in order to execute its function, the tubby family of proteins would functionally interact with cargo, the periciliary membrane components and PIP2 rich lipids. In further support of this hypothesis, we have now shown that LOC69239 interacts with USH2 complex at the periciliary membrane in photoreceptors. We made progress in the past year by identifying LOC69239 as a tubby interacting protein and shown that it is expressed specifically in photoreceptor cells. It localizes along the ciliary rootlets. In collaboration with Lijin Dong at the GEC we have ablated its expression using CRISPR technology in mice and we are analyzing its function and disease mechanism (Vetrivel Sengottuvel). 5. Mammalian Prickle homologs and planar cell polarity. Through genetic ablation studies, we show that Prickle 1 (Pk1) is a major player for tissue-axis extension and organization. In Pk1 mutant mice, the tissue-axis defects are seen in both appendages and internal organs including heart and kidney with severely disorganized cell arrangement, aberrant ciliogenesis, mislocalization of polarity proteins such as Vangl and Dvl and actin machinery. Cultured mouse embryonic fibroblast cells (MEF) of the mutant embryos show a prolonged wound-healing process in cell scratch assay, and recapitulate some of the cellular defects in tissues including actin polymerization and aberrant ciliogenesis. Pk1-directed tissue extension is a systematic event at all tissue levels throughout inner ear cochlea, axonal innervation and blood vasculature. (Chunqiao Liu et al).
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