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Pietro De Camilli - One of the best experts on this subject based on the ideXlab platform.
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OCRL interactors identified by label-free quantitative proteomics from cell lines expressing GFP-OCRL near endogenous levels
2016Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, Pietro De CamilliAbstract:OCRL interactors identified by label-free quantitative proteomics from cell lines expressing GFP-OCRL near endogenous level
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OCRL interactors identified by label-free quantitative proteomics in immunoprecipitates from cell lines expressing GFP-OCRL at 5X endogenous levels
2016Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, Pietro De CamilliAbstract:OCRL interactors identified by label-free quantitative proteomics in immunoprecipitates from cell lines expressing GFP-OCRL at 5X endogenous level
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a role of OCRL in clathrin coated pit dynamics and uncoating revealed by studies of lowe syndrome cells
eLife, 2014Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, James S Duncan, Liang Liang, Pietro De CamilliAbstract:Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations. DOI: http://dx.doi.org/10.7554/eLife.02975.001
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inositol 5 phosphatases insights from the lowe syndrome protein OCRL
Trends in Biochemical Sciences, 2012Co-Authors: Michelle Pirruccello, Pietro De CamilliAbstract:The precise regulation of phosphoinositide lipids in cellular membranes is crucial for cellular survival and function. Inositol 5-phosphatases have been implicated in a variety of disorders, including various cancers, obesity, type 2 diabetes, neurodegenerative diseases and rare genetic conditions. Despite the obvious impact on human health, relatively little structural and biochemical information is available for this family. Here, we review recent structural and mechanistic work on the 5-phosphatases with a focus on OCRL, whose loss of function results in oculocerebrorenal syndrome of Lowe and Dent 2 disease. Studies of OCRL emphasize how the actions of 5-phosphatases rely on both intrinsic and extrinsic membrane recognition properties for full catalytic function. Additionally, structural analysis of missense mutations in the catalytic domain of OCRL provides insight into the phenotypic heterogeneity observed in Lowe syndrome and Dent disease.
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recognition of the f h motif by the lowe syndrome protein OCRL
Nature Structural & Molecular Biology, 2011Co-Authors: Michelle Pirruccello, Laura E. Swan, Ewa Foltastogniew, Pietro De CamilliAbstract:The protein OCRL is linked to Lowe syndrome and Dent disease, two related diseases. Mutations in the OCRL Ash-RhoGAP domain abolish its interactions with the F&H motifs in APPL1, Ses1 and Ses2. Structural and biochemical analysis of the OCRL Ash-RhoGAP domain with F&H motifs shows that clinical mutations destabilize the Ash-RhoGAP domain, abolishing the interactions between OCRL- and F&H-motif-containing proteins.
Robert L Nussbaum - One of the best experts on this subject based on the ideXlab platform.
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kidney tubular ablation of OCRL inpp5b phenocopies lowe syndrome tubulopathy
Journal of The American Society of Nephrology, 2017Co-Authors: Kazunori Inoue, Ramiro Nandez, Daniel M Balkin, Robert L Nussbaum, Lijuan Liu, Xuefei Tian, Tong WangAbstract:Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of OCRL in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global OCRL-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.
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mouse model for lowe syndrome dent disease 2 renal tubulopathy
Journal of The American Society of Nephrology, 2011Co-Authors: Susan P Bothwell, Emily Chan, William A. Gahl, Isa Bernardini, Robert L NussbaumAbstract:The Lowe oculocerebrorenal syndrome is an X-linked disorder characterized by congenital cataracts, cognitive disability, and proximal tubular dysfunction. Both this syndrome and Dent Disease 2 result from loss-of-function mutations in the OCRL gene, which encodes a type II phosphatidylinositol bisphosphate 5-phosphatase. OCRL-deficient mice are unaffected, however, which we believe reflects a difference in how humans and mice cope with the enzyme deficiency. Inpp5b and INPP5B, paralogous autosomal genes that encode another type II phosphoinositide 5-phosphatase in mice and humans, respectively, might explain the distinct phenotype in the two species because they are the closest paralogs to OCRL and OCRL in their respective genomes yet differ between the two species with regard to expression and splicing. Here, we generated OCRL(-/-) mice that express INPP5B but not Inpp5b. Similar to the human syndromes, all showed reduced postnatal growth, low molecular weight proteinuria, and aminoaciduria. Thus, we created an animal model for OCRL and Dent Disease 2 tubulopathy by humanizing a modifier paralog in mice already carrying the mutant disease gene.
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Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome
Mammalian Genome, 2010Co-Authors: Susan P Bothwell, Leslie W. Farber, Adam Hoagland, Robert L NussbaumAbstract:The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes OCRL, a type II phosphatidylinositol bisphosphate (PtdIns4,5P_2) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog OCRL have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between OCRL-deficient humans and mice was likely due to differences in how the two species compensate for loss of the OCRL enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P_2 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b / INPP5B are the most highly conserved paralogs to OCRL / OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with OCRL in mice in vivo . We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B . We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of OCRL function and, by providing insight into the cellular roles of OCRL and Inpp5b, aid in the development of a model system in which to study Lowe syndrome.
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x inactivation analysis of embryonic lethality in OCRL wt inpp5b mice
Mammalian Genome, 2010Co-Authors: David J Bernard, Robert L NussbaumAbstract:Mutations in the human OCRL gene, which encodes a phosphatidylinositol(4,5)bisphosphate 5-phosphatase, result in the X-linked oculocerebrorenal syndrome of Lowe. Mice with a targeted disruption of OCRL have no phenotypic abnormalities. Targeted disruption of its closest paralog, Inpp5b, causes male infertility in the 129S6 background. Mice with disruptions of both genes are lost in utero prior to 9.5-10.5 dpc, indicating that there is a functional overlap between the two paralogs early in development. We analyzed the pattern of X-inactivation in four tissues of distinct embryonic origin from OCRLwt/−;Inpp5b−/− females to explore the timing and tissue distribution of the functional overlap. X-inactivation was strongly skewed against the disrupted OCRL− allele being on the active X chromosome in all four tissues tested, indicating that there is early selection against cell lineages lacking both OCRL and Inpp5b. Extraembryonic tissue was also involved in the lethality because there were never any live-born OCRLwt/−;Inpp5b−/− females when the functional OCRLwt allele was on the paternal X chromosome, which is preferentially inactivated in trophoblast-derived extraembryonic tissues. Live-born OCRLwt/−;Inpp5b−/− females were found when the functional OCRLwt allele was maternal, although in fewer numbers than expected. The importance of the extraembryonic tissues in the early embryonic lethality of embryos lacking both OCRL and Inpp5b is reinforced by the successful isolation of a viable 40,XX OCRL−/−;Inpp5b−/− embryonic stem cell from the inner cell mass of a 3.5-dpc blastocyst prior to implantation. These results indicate a functional overlap of OCRL and Inpp5b in most cell lineages, especially in extraembryonic tissues.
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spectrum of mutations in the OCRL1 gene in the lowe oculocerebrorenal syndrome
American Journal of Human Genetics, 1997Co-Authors: Ti Lin, Richard A. Lewis, Annmarie Leahey, Bonnie M Orrison, Sharon F Suchy, David J Bernard, Robert L NussbaumAbstract:The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder characterized by congenital cataracts, mental retardation, and renal Fanconi syndrome. The OCRL1 gene, which, when mutated, is responsible for OCRL, encodes a 105-kD Golgi protein with phosphatidylinositol (4,5)bisphosphate (PtdIn[4,5]P2) 5-phosphatase activity. We have examined the OCRL1 gene in 12 independent patients with OCRL and have found 11 different mutations. Six were nonsense mutations, and one a deletion of one or two nucleotides that leads to frameshift and premature termination. In one, a 1.2-kb genomic deletion of exon 14 was identified. In four others, missense mutations or the deletion of a single codon were found to involve amino acid residues known to be highly conserved among proteins with PtdIns(4,5)P2 5-phosphatase activity. All patients had markedly reduced PtdIns(4,5)P2 5-phosphatase activity in their fibroblasts, whereas the OCRL1 protein was detectable by immunoblotting in some patients with either missense mutations or a codon deletion but was not detectable in those with premature termination mutations. These results confirm and extend our previous observation that the OCRL phenotype results from loss of function of the OCRL1 protein and that mutations are generally heterogeneous. Missense mutations that abolish enzyme activity but not expression of the protein will be useful for studying structure-function relationships in PtdIns(4,5)P2 5-phosphatases.
Yang Sun - One of the best experts on this subject based on the ideXlab platform.
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OCRL regulates lysosome positioning and mtorc1 activity through ssx2ip mediated microtubule anchoring
EMBO Reports, 2021Co-Authors: Biao Wang, Jorge A Alvarado, Yang Sun, Philipp P Prosseda, Tia J Kowal, Qing WangAbstract:Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule-anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule-organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule-organizing centers and impaired microtubule-based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome-targeted PACT-SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients' cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.
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loss of OCRL increases ciliary pi 4 5 p2 in lowe oculocerebrorenal syndrome
PMC, 2017Co-Authors: Philipp P Prosseda, Na Luo, Jorge A Alvarado, Yang Sun, Biao WangAbstract:ABSTRACT Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts and glaucoma, mental retardation, and proximal renal tubular dysfunction. Mutations in OCRL, an inositol polyphosphate 5-phosphatase that dephosphorylates PI(4,5)P 2 , cause Lowe syndrome. Previously we showed that OCRL localizes to the primary cilium, which has a distinct membrane phospholipid composition, but disruption of phosphoinositides in the ciliary membrane is poorly understood. Here, we demonstrate that cilia from Lowe syndrome patient fibroblasts exhibit increased levels of PI(4,5)P 2 and decreased levels of PI4P. In particular, subcellular distribution of PI(4,5)P 2 build-up was observed at the transition zone. Accumulation of ciliary PI(4,5)P 2 was pronounced in mouse embryonic fibroblasts (MEFs) derived from Lowe syndrome mouse model as well as in OCRL -null MEFs, which was reversed by reintroduction of OCRL. Similarly, expression of wild-type OCRL reversed the elevated PI(4,5)P 2 in Lowe patient cells. Accumulation of sonic hedgehog protein in response to hedgehog agonist was decreased in MEFs derived from a Lowe syndrome mouse model. Together, our findings show for the first time an abnormality in ciliary phosphoinositides of both human and mouse cell models of Lowe syndrome.
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compensatory role of inositol 5 phosphatase inpp5b to OCRL in primary cilia formation in oculocerebrorenal syndrome of lowe
PLOS ONE, 2013Co-Authors: Na Luo, Michael Conwell, Robert N. Weinreb, Akhilesh Kumar, Ryan Anderson, Yang SunAbstract:Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm’s canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer’s vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.
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OCRL localizes to the primary cilium a new role for cilia in lowe syndrome
Human Molecular Genetics, 2012Co-Authors: Na Luo, Clark D. Wells, Robert N. Weinreb, Callah West, Carlos Murgazamalloa, Lou Sun, Ryan M Anderson, Jeffrey B Travers, Hemant Khanna, Yang SunAbstract:Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of OCRL in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.
Olivier Devuyst - One of the best experts on this subject based on the ideXlab platform.
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The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease
Kidney international, 2020Co-Authors: Marine Berquez, Beatrice Paola Festa, Olivier Devuyst, Alessandro Luciani, Jonathan R. Gadsby, Richard J. Butler, Stephen P. Jackson, Valeria Berno, Jennifer L. GallopAbstract:Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OCRLY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.
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the 5 phosphatase OCRL in lowe syndrome and dent disease 2
Nature Reviews Nephrology, 2017Co-Authors: Maria Antonietta De Matteis, Leopoldo Staiano, Francesco Emma, Olivier DevuystAbstract:Dysfunction of endolysosomal pathways can lead to generalized dysfunction of the proximal tubule. Here, De Matteis and colleagues describe the role of the inositol polyphosphate 5-phosphatase, OCRL, in the endolysosomal pathway and how mutations in the encoding gene lead to the clinical manifestations of Lowe syndrome and Dent disease 2. Lowe syndrome is an X-linked disease that is characterized by congenital cataracts, central hypotonia, intellectual disability and renal Fanconi syndrome. The disease is caused by mutations in OCRL, which encodes an inositol polyphosphate 5-phosphatase (OCRL) that acts on phosphoinositides — quantitatively minor constituents of cell membranes that are nonetheless pivotal regulators of intracellular trafficking. In this Review we summarize the considerable progress made over the past decade in understanding the cellular roles of OCRL in regulating phosphoinositide balance along the endolysosomal pathway, a fundamental system for the reabsorption of proteins and solutes by proximal tubular cells. We discuss how studies of OCRL have led to important discoveries about the basic mechanisms of membrane trafficking and describe the key features and limitations of the currently available animal models of Lowe syndrome. Mutations in OCRL can also give rise to a milder pathology, Dent disease 2, which is characterized by renal Fanconi syndrome in the absence of extrarenal pathologies. Understanding how mutations in OCRL give rise to two clinical entities with differing extrarenal manifestations represents an opportunity to identify molecular pathways that could be targeted to develop treatments for these conditions.
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the 5 phosphatase OCRL in lowe syndrome and dent disease 2
Nature Reviews Nephrology, 2017Co-Authors: Maria Antonietta De Matteis, Leopoldo Staiano, Francesco Emma, Olivier DevuystAbstract:Lowe syndrome is an X-linked disease that is characterized by congenital cataracts, central hypotonia, intellectual disability and renal Fanconi syndrome. The disease is caused by mutations in OCRL, which encodes an inositol polyphosphate 5-phosphatase (OCRL) that acts on phosphoinositides - quantitatively minor constituents of cell membranes that are nonetheless pivotal regulators of intracellular trafficking. In this Review we summarize the considerable progress made over the past decade in understanding the cellular roles of OCRL in regulating phosphoinositide balance along the endolysosomal pathway, a fundamental system for the reabsorption of proteins and solutes by proximal tubular cells. We discuss how studies of OCRL have led to important discoveries about the basic mechanisms of membrane trafficking and describe the key features and limitations of the currently available animal models of Lowe syndrome. Mutations in OCRL can also give rise to a milder pathology, Dent disease 2, which is characterized by renal Fanconi syndrome in the absence of extrarenal pathologies. Understanding how mutations in OCRL give rise to two clinical entities with differing extrarenal manifestations represents an opportunity to identify molecular pathways that could be targeted to develop treatments for these conditions.
Ramiro Nandez - One of the best experts on this subject based on the ideXlab platform.
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kidney tubular ablation of OCRL inpp5b phenocopies lowe syndrome tubulopathy
Journal of The American Society of Nephrology, 2017Co-Authors: Kazunori Inoue, Ramiro Nandez, Daniel M Balkin, Robert L Nussbaum, Lijuan Liu, Xuefei Tian, Tong WangAbstract:Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of OCRL in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global OCRL-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.
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OCRL interactors identified by label-free quantitative proteomics from cell lines expressing GFP-OCRL near endogenous levels
2016Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, Pietro De CamilliAbstract:OCRL interactors identified by label-free quantitative proteomics from cell lines expressing GFP-OCRL near endogenous level
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OCRL interactors identified by label-free quantitative proteomics in immunoprecipitates from cell lines expressing GFP-OCRL at 5X endogenous levels
2016Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, Pietro De CamilliAbstract:OCRL interactors identified by label-free quantitative proteomics in immunoprecipitates from cell lines expressing GFP-OCRL at 5X endogenous level
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a role of OCRL in clathrin coated pit dynamics and uncoating revealed by studies of lowe syndrome cells
eLife, 2014Co-Authors: Ramiro Nandez, Summer Paradise, Mirko Messa, Heather Czapla, Daniel M Balkin, Marco Y Hein, Matthias Mann, James S Duncan, Liang Liang, Pietro De CamilliAbstract:Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations. DOI: http://dx.doi.org/10.7554/eLife.02975.001