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The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule.

Oltrabella F, Pietka G, Ramirez IB, Mironov A, Starborg T, Drummond IA, Hinchliffe KA, Lowe M - PLoS Genet. (2015)

Bottom Line: This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway.We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells.Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.

No MeSH data available.


Related in: MedlinePlus

Pronephric cilia in ocrl-/- zebrafish.A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, ocrl-/- mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, ocrl-/- right). Dextran excretion was identical in control and ocrl-/- embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows ocrl-/- mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), ocrl-/- mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and double bubble (dbb) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).
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pgen.1005058.g005: Pronephric cilia in ocrl-/- zebrafish.A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, ocrl-/- mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, ocrl-/- right). Dextran excretion was identical in control and ocrl-/- embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows ocrl-/- mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), ocrl-/- mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and double bubble (dbb) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).

Mentions: Several studies have described defects in ciliogenesis upon OCRL1 depletion, and proposed that the symptoms of Lowe syndrome and Dent-2 disease are due to ciliary defects [16,17,25]. It has also recently been reported that loss of primary cilia in the renal tubule can reduce the rate of apical endocytosis due to impaired detection of fluid shear stress [44]. We therefore investigated whether effects upon ciliary function could account for endocytic defects we observe in the OCRL1 deficient embryos, possibly as a consequence of impaired fluid flow within the pronephric tubule. As previously reported, OCRL1-deficient embryos had shorter and fewer cilia, labelled using an antibody to acetylated tubulin, within the pronephros [16] (Fig. 5A), consistent with a role for OCRL1 in ciliogenesis. However, several lines of evidence indicate that impaired ciliary function cannot account for the endocytic defect observed in the OCRL1 deficient embryos. Firstly, we do not observe significant impairment of fluid flow within the pronephros of ocrl-/- embryos, as indicated by normal excretion of unabsorbed fluorescent dextran from the cloacae (Fig. 5B). In line with this, the mutant does not develop renal cysts (Fig. 5C). Secondly, we compared endocytosis in the ocrl-/- mutant with embryos injected with a morpholino against IFT88/polaris, which is required for ciliogenesis in the pronephric tubule [45]. Embryos injected with high doses of morpholino displayed characteristic features of defective ciliogenesis, with a curved body axis, oedema and dilation of the renal tubule (Fig. 5C). Notably, these phenotypes are absent from the ocrl-/- embryos (Fig. 5C). Consistent with the study of [44], renal endocytosis was impaired in the IFT88/polaris morphants. In embryos injected with lower doses of morpholino, a ciliogenesis defect was still evident, which was comparable to (at 2 ng morpholino) or more severe than (at 4 ng morpholino) that observed in the ocrl-/- mutant (Fig. 5D). Importantly, these IFT88/polaris morphants were still able to carry out endocytosis, indicating that the impaired endocytosis in the ocrl-/- mutant cannot be explained by a defect in ciliogenesis (Fig. 5E). Further support comes from the observation that the zebrafish cilia mutant double bubble, which also has a more severe ciliogenesis defect than the ocrl-/- line, can endocytose dextran [32,46] (Fig. 5F). Our results indicate that defects in dextran uptake and ciliogenesis are separable, and that loss of endocytic tracer uptake in the OCRL1 mutant and morphant embryos is not a downstream consequence of impaired ciliary function.


The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule.

Oltrabella F, Pietka G, Ramirez IB, Mironov A, Starborg T, Drummond IA, Hinchliffe KA, Lowe M - PLoS Genet. (2015)

Pronephric cilia in ocrl-/- zebrafish.A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, ocrl-/- mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, ocrl-/- right). Dextran excretion was identical in control and ocrl-/- embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows ocrl-/- mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), ocrl-/- mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and double bubble (dbb) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).
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Related In: Results  -  Collection

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pgen.1005058.g005: Pronephric cilia in ocrl-/- zebrafish.A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, ocrl-/- mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, ocrl-/- right). Dextran excretion was identical in control and ocrl-/- embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows ocrl-/- mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), ocrl-/- mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), ocrl-/- mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and double bubble (dbb) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).
Mentions: Several studies have described defects in ciliogenesis upon OCRL1 depletion, and proposed that the symptoms of Lowe syndrome and Dent-2 disease are due to ciliary defects [16,17,25]. It has also recently been reported that loss of primary cilia in the renal tubule can reduce the rate of apical endocytosis due to impaired detection of fluid shear stress [44]. We therefore investigated whether effects upon ciliary function could account for endocytic defects we observe in the OCRL1 deficient embryos, possibly as a consequence of impaired fluid flow within the pronephric tubule. As previously reported, OCRL1-deficient embryos had shorter and fewer cilia, labelled using an antibody to acetylated tubulin, within the pronephros [16] (Fig. 5A), consistent with a role for OCRL1 in ciliogenesis. However, several lines of evidence indicate that impaired ciliary function cannot account for the endocytic defect observed in the OCRL1 deficient embryos. Firstly, we do not observe significant impairment of fluid flow within the pronephros of ocrl-/- embryos, as indicated by normal excretion of unabsorbed fluorescent dextran from the cloacae (Fig. 5B). In line with this, the mutant does not develop renal cysts (Fig. 5C). Secondly, we compared endocytosis in the ocrl-/- mutant with embryos injected with a morpholino against IFT88/polaris, which is required for ciliogenesis in the pronephric tubule [45]. Embryos injected with high doses of morpholino displayed characteristic features of defective ciliogenesis, with a curved body axis, oedema and dilation of the renal tubule (Fig. 5C). Notably, these phenotypes are absent from the ocrl-/- embryos (Fig. 5C). Consistent with the study of [44], renal endocytosis was impaired in the IFT88/polaris morphants. In embryos injected with lower doses of morpholino, a ciliogenesis defect was still evident, which was comparable to (at 2 ng morpholino) or more severe than (at 4 ng morpholino) that observed in the ocrl-/- mutant (Fig. 5D). Importantly, these IFT88/polaris morphants were still able to carry out endocytosis, indicating that the impaired endocytosis in the ocrl-/- mutant cannot be explained by a defect in ciliogenesis (Fig. 5E). Further support comes from the observation that the zebrafish cilia mutant double bubble, which also has a more severe ciliogenesis defect than the ocrl-/- line, can endocytose dextran [32,46] (Fig. 5F). Our results indicate that defects in dextran uptake and ciliogenesis are separable, and that loss of endocytic tracer uptake in the OCRL1 mutant and morphant embryos is not a downstream consequence of impaired ciliary function.

Bottom Line: This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway.We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells.Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.

No MeSH data available.


Related in: MedlinePlus