1. Post-translational modification of RPGR, TTLL5 and disease mechanisms. Mutations in the X-linked RPGR gene are a major cause of retinitis pigmentosa. ORF15 variant of RPGR (RPGRORF15), carrying multiple Glu-Gly tandem repeats and a C-terminal basic domain of unknown function, localizes to the connecting cilium where it is thought to regulate cargo trafficking. In a recently completed study, we show that tubulin tyrosine ligase like-5 (TTLL5) glutamylates RPGRORF15 in its Glu-Gly rich repetitive region containing motifs homologous to the -tubulin C-terminal tail. The RPGRORF15 C-terminal basic domain binds to the non-catalytic cofactor interaction domain unique to TTLL5 among TTLL family glutamylases, and targets TTLL5 to glutamylate RPGR. Only TTLL5 and not other TTLL family glutamylases, interacts with RPGRORF15 when expressed transiently in cells. Consistent with this, a Ttll5 mutant mouse displays a complete loss of RPGR glutamylation without marked changes in tubulin glutamylation levels. The Ttll5 mutant mouse develops slow photoreceptor degeneration with early mislocalization of cone opsins, features resembling those of Rpgr null mice. Moreover TTLL5 disease mutants that cause human retinal dystrophy show impaired glutamylation of RPGRORF15. Thus RPGRORF15 is a novel glutamylation substrate, and this post-translational modification is critical for its function in photoreceptors. Our study uncovers the pathogenic mechanism whereby absence of RPGRORF15 glutamylation leads to retinal pathology in patients with TTLL5 gene mutations and connects these two genes into a common disease pathway. A manuscript describing this work has been published (Xun et al.). Following completion of this study, we have begun to ask more in depth questions about the role of RPGR post-translational modifications. Based on the model of tubulin modification, we propose that the glutamylated region of RPGR-ORF15 may serve to recruit additional factors to the ciliary transition zone. We carried out a differential protein interaction screens. Using WT and ttll5 null mice, we performed co-immunoprecipitations followed by mass spectrometry and identified several interesting candidates. The rationale of these experiments is that RPGR in WWT retina will recruit additional protein partners, whereas RPGR in ttll5 mutant retina is not glutamylated and therefore unable to bind those additional interaction partners (negative control). In addition we have also initiated a gene therapy study of TTLL5 in collaboration with Dr. Zhijian Wus group. 2. Loss of Macf1 abolishes ciliogenesis and disrupts apicobasal polarity establishment in the retina. 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. A manuscript has been accepted for publication (Helen May-Simera et al). 3. Stem cell studies. Our long-term goal is to make stem cell derived photoreceptors that are well differentiated as judged but the elaboration of outer segments. Such a culture system could be used for disease mechanism studies, drug discoveries and provide donor tissues for transplantation. Given the dependence of photoreceptors on RPE, the latter is also a part of the studies. One of the critical components that appears to missing in current 3D retinal culture systems is a functioning RPE layer. We have used an embryonic stem (ES) cell line (H9) to differentiate stem cells into RPE and after 60 days of induction we have been able to confirm successful differentiation of the ES cells into RPE cells. Our differentiated RPE cells correctly display RPE markers, express typical proteins of mature RPE cells, have polarity of mature RPE cells, and display distinct RPE morphology such as: forming necessary tight junctions between the cells required for epithelial monolayer and hexagonal shape. This shows reliability and success of our protocol and allows a baseline comparison of differentiation of Induced Pluripotent Stem Cells (iPSCs). We have begun experiments to differentiate iPSCs from patients into RPE are underway. These experiments show that the iPSCs are correctly developing into RPE at D45 so far with all of the same phenotypes as the ES cells that have been differentiated, although the process seems to be delayed slightly in iPSCs. We have successfully differentiated ES and iPSCs into RPE cells on a Matrigel coating. These efforts are spearheaded by Dr. Dawn Landis in our Section in collaboration with Dr. Swaroops Stem Cell group. 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. We have made substantial progress in characterizing the function of LOC69239; We show that it is required for photoreceptor viability in mice and in zebrafish, and propose that this protein functions in endosomal trafficking. A manuscript describing this work is in preparation (Dr. Vetrivel Sengottuvel).
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