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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

Rescue of the pronephric uptake defect in OCRL1 deficient embryos.A. Lateral view of a wild-type zebrafish embryo co-injected with cmlc2:GFP and enpep:OCRL1a. B. Schematic diagram of OCRL1 showing domain organization and localization of deletion and point mutants used in rescue experiments. C. RT-PCR detection of OCRL1 mRNA in WT, ocrl-/- embryos or ocrl-/- embryos expressing the indicated OCRL1a constructs. eIF1α was used as a control. The vector only lane is a positive control for the OCRL1 PCR, and lacks eIF1α. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT), ocrl-/- embryos or ocrl-/- embryos expressing OCRL1a, catalytically inactive GFP-OCRL1a (D480A), OCRL1a unable to bind clathrin (ΔLIDID ΔLIDLG), Rab GTPases (F727V), F&H domain proteins such as APPL1 and IPIP27A/B (W798A), or OCRL1a harbouring the Lowe syndrome mutation L746P or Dent-2 mutation P858L. The pronephric tubules are indicated with a dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. 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.
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pgen.1005058.g006: Rescue of the pronephric uptake defect in OCRL1 deficient embryos.A. Lateral view of a wild-type zebrafish embryo co-injected with cmlc2:GFP and enpep:OCRL1a. B. Schematic diagram of OCRL1 showing domain organization and localization of deletion and point mutants used in rescue experiments. C. RT-PCR detection of OCRL1 mRNA in WT, ocrl-/- embryos or ocrl-/- embryos expressing the indicated OCRL1a constructs. eIF1α was used as a control. The vector only lane is a positive control for the OCRL1 PCR, and lacks eIF1α. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT), ocrl-/- embryos or ocrl-/- embryos expressing OCRL1a, catalytically inactive GFP-OCRL1a (D480A), OCRL1a unable to bind clathrin (ΔLIDID ΔLIDLG), Rab GTPases (F727V), F&H domain proteins such as APPL1 and IPIP27A/B (W798A), or OCRL1a harbouring the Lowe syndrome mutation L746P or Dent-2 mutation P858L. The pronephric tubules are indicated with a dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. 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.

Mentions: To confirm the specificity of the renal endocytosis defect, and to dissect the mechanisms involved, we performed rescue experiments in which OCRL1 was transiently expressed in mutant embryos under the control of the enpep kidney-specific promoter [35]. We used untagged zebrafish OCRL1 for these experiments. In order to identify transduced embryos, we co-expressed GFP under the control of the cardiac myosin light chain 2 (cmlc2) promoter, which strongly labels the heart (Fig. 6A). Control experiments indicated that co-expression was efficient, with 75% of embryos expressing the cmlc2-GFP reporter also positive for enpep-GFP in the pronephros. In the case of OCRL1 (domain organization shown in Fig. 6B), we could confirm expression by RT-PCR (Fig. 6C). As shown in Fig. 6D, re-expression of OCRL1 in the pronephros of ocrl-/- embryos was able to restore dextran endocytosis. Quantitation revealed that of the cmlc2-GFP positive embryos co-expressing wild-type OCRL1, 60% had efficient (normal) dextran uptake, with an additional 35% displaying a lower degree of uptake (Fig. 6E). This compares favourably to wild-type embryos which have 80–90% high uptake, indicating an efficient rescue.


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)

Rescue of the pronephric uptake defect in OCRL1 deficient embryos.A. Lateral view of a wild-type zebrafish embryo co-injected with cmlc2:GFP and enpep:OCRL1a. B. Schematic diagram of OCRL1 showing domain organization and localization of deletion and point mutants used in rescue experiments. C. RT-PCR detection of OCRL1 mRNA in WT, ocrl-/- embryos or ocrl-/- embryos expressing the indicated OCRL1a constructs. eIF1α was used as a control. The vector only lane is a positive control for the OCRL1 PCR, and lacks eIF1α. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT), ocrl-/- embryos or ocrl-/- embryos expressing OCRL1a, catalytically inactive GFP-OCRL1a (D480A), OCRL1a unable to bind clathrin (ΔLIDID ΔLIDLG), Rab GTPases (F727V), F&H domain proteins such as APPL1 and IPIP27A/B (W798A), or OCRL1a harbouring the Lowe syndrome mutation L746P or Dent-2 mutation P858L. The pronephric tubules are indicated with a dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. 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.
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pgen.1005058.g006: Rescue of the pronephric uptake defect in OCRL1 deficient embryos.A. Lateral view of a wild-type zebrafish embryo co-injected with cmlc2:GFP and enpep:OCRL1a. B. Schematic diagram of OCRL1 showing domain organization and localization of deletion and point mutants used in rescue experiments. C. RT-PCR detection of OCRL1 mRNA in WT, ocrl-/- embryos or ocrl-/- embryos expressing the indicated OCRL1a constructs. eIF1α was used as a control. The vector only lane is a positive control for the OCRL1 PCR, and lacks eIF1α. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT), ocrl-/- embryos or ocrl-/- embryos expressing OCRL1a, catalytically inactive GFP-OCRL1a (D480A), OCRL1a unable to bind clathrin (ΔLIDID ΔLIDLG), Rab GTPases (F727V), F&H domain proteins such as APPL1 and IPIP27A/B (W798A), or OCRL1a harbouring the Lowe syndrome mutation L746P or Dent-2 mutation P858L. The pronephric tubules are indicated with a dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. 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.
Mentions: To confirm the specificity of the renal endocytosis defect, and to dissect the mechanisms involved, we performed rescue experiments in which OCRL1 was transiently expressed in mutant embryos under the control of the enpep kidney-specific promoter [35]. We used untagged zebrafish OCRL1 for these experiments. In order to identify transduced embryos, we co-expressed GFP under the control of the cardiac myosin light chain 2 (cmlc2) promoter, which strongly labels the heart (Fig. 6A). Control experiments indicated that co-expression was efficient, with 75% of embryos expressing the cmlc2-GFP reporter also positive for enpep-GFP in the pronephros. In the case of OCRL1 (domain organization shown in Fig. 6B), we could confirm expression by RT-PCR (Fig. 6C). As shown in Fig. 6D, re-expression of OCRL1 in the pronephros of ocrl-/- embryos was able to restore dextran endocytosis. Quantitation revealed that of the cmlc2-GFP positive embryos co-expressing wild-type OCRL1, 60% had efficient (normal) dextran uptake, with an additional 35% displaying a lower degree of uptake (Fig. 6E). This compares favourably to wild-type embryos which have 80–90% high uptake, indicating an efficient rescue.

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