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Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.

Ying H, Shen X, Park B, Yue BY - PLoS ONE (2010)

Bottom Line: It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein.Treatment of nocadazole resulted in dispersion of the optineurin foci.The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.

Methodology/principal findings: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP) fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time.

Conclusions/significance: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.

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Related in: MedlinePlus

Cellular localization of overexpressed wild type optineurin.A. RGC5 cells were transfected for 20 h, fixed and immunostained. When transfected with pEGFP-N1 (mock control, top, left panel), green fluorescence was observed in the entire RGC5 cells including the nucleus. When transfected with pOPTN-EGFP (OPTN-GFP, bottom, left panel), diffuse green fluorescence was likewise seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures termed foci were also noted in the perinuclear region around the Golgi apparatus (stained with anti-GM130 in red). Insets show the enlarged and merged images in the perinuclear region. Optineurin foci largely did not overlap with the GM130 staining. Golgi fragmentation in pOPTN-EGFP-transfected cells (arrows) was observed. Scale bar, 10 µm. B. RGC5 cells were transfected with pOPTN-EGFP for 20 h and underwent differentiation with treatment of 316 nM staurosporine for 4 h. Punctate optineurin-GFP foci (green) were found on the neurite extensions (arrowheads). The enlarged and amplified image of the blocked area is shown in the right panel. Scale bars, 10 µm.
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pone-0009168-g003: Cellular localization of overexpressed wild type optineurin.A. RGC5 cells were transfected for 20 h, fixed and immunostained. When transfected with pEGFP-N1 (mock control, top, left panel), green fluorescence was observed in the entire RGC5 cells including the nucleus. When transfected with pOPTN-EGFP (OPTN-GFP, bottom, left panel), diffuse green fluorescence was likewise seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures termed foci were also noted in the perinuclear region around the Golgi apparatus (stained with anti-GM130 in red). Insets show the enlarged and merged images in the perinuclear region. Optineurin foci largely did not overlap with the GM130 staining. Golgi fragmentation in pOPTN-EGFP-transfected cells (arrows) was observed. Scale bar, 10 µm. B. RGC5 cells were transfected with pOPTN-EGFP for 20 h and underwent differentiation with treatment of 316 nM staurosporine for 4 h. Punctate optineurin-GFP foci (green) were found on the neurite extensions (arrowheads). The enlarged and amplified image of the blocked area is shown in the right panel. Scale bars, 10 µm.

Mentions: When transfected with pEGFP-N1 (mock control), fluorescence from the expressed green fluorescence protein (GFP) was observed in the entire RGC5 cells including the nucleus (Fig. 3A, top left panel). When transfected with pOPTN-EGFP plasmid, diffuse green fluorescence from optineurin-GFP fusion protein was also seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures in the perinuclear regions were noted (Fig. 3A, bottom left panel). These structures were very similar to those termed foci found previously in RPE cells [17]. Live cell imaging indicated that the foci were not stationary. They fused with or separated from each other (Video S1) and were mobile, sometimes exhibiting long distance movements (Video S2). When immunostained with Golgi marker GM130 in red (Fig. 3A, right panels), the optineurin foci in RGC5 cells did not colocalize with the Golgi apparatus (Fig. 3A, insert, bottom right panel). The lack of major colocalization, confirmed by confocal microscopy after sequential scanning, was also demonstrated previously in human RPE and trabecular meshwork cells [17]. Golgi fragmentation (Fig. 3A, arrow) was in addition noted in all these cell types [17].


Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.

Ying H, Shen X, Park B, Yue BY - PLoS ONE (2010)

Cellular localization of overexpressed wild type optineurin.A. RGC5 cells were transfected for 20 h, fixed and immunostained. When transfected with pEGFP-N1 (mock control, top, left panel), green fluorescence was observed in the entire RGC5 cells including the nucleus. When transfected with pOPTN-EGFP (OPTN-GFP, bottom, left panel), diffuse green fluorescence was likewise seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures termed foci were also noted in the perinuclear region around the Golgi apparatus (stained with anti-GM130 in red). Insets show the enlarged and merged images in the perinuclear region. Optineurin foci largely did not overlap with the GM130 staining. Golgi fragmentation in pOPTN-EGFP-transfected cells (arrows) was observed. Scale bar, 10 µm. B. RGC5 cells were transfected with pOPTN-EGFP for 20 h and underwent differentiation with treatment of 316 nM staurosporine for 4 h. Punctate optineurin-GFP foci (green) were found on the neurite extensions (arrowheads). The enlarged and amplified image of the blocked area is shown in the right panel. Scale bars, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820081&req=5

pone-0009168-g003: Cellular localization of overexpressed wild type optineurin.A. RGC5 cells were transfected for 20 h, fixed and immunostained. When transfected with pEGFP-N1 (mock control, top, left panel), green fluorescence was observed in the entire RGC5 cells including the nucleus. When transfected with pOPTN-EGFP (OPTN-GFP, bottom, left panel), diffuse green fluorescence was likewise seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures termed foci were also noted in the perinuclear region around the Golgi apparatus (stained with anti-GM130 in red). Insets show the enlarged and merged images in the perinuclear region. Optineurin foci largely did not overlap with the GM130 staining. Golgi fragmentation in pOPTN-EGFP-transfected cells (arrows) was observed. Scale bar, 10 µm. B. RGC5 cells were transfected with pOPTN-EGFP for 20 h and underwent differentiation with treatment of 316 nM staurosporine for 4 h. Punctate optineurin-GFP foci (green) were found on the neurite extensions (arrowheads). The enlarged and amplified image of the blocked area is shown in the right panel. Scale bars, 10 µm.
Mentions: When transfected with pEGFP-N1 (mock control), fluorescence from the expressed green fluorescence protein (GFP) was observed in the entire RGC5 cells including the nucleus (Fig. 3A, top left panel). When transfected with pOPTN-EGFP plasmid, diffuse green fluorescence from optineurin-GFP fusion protein was also seen in the cytoplasm of RGC5 cells. In addition, bright granular or punctuate structures in the perinuclear regions were noted (Fig. 3A, bottom left panel). These structures were very similar to those termed foci found previously in RPE cells [17]. Live cell imaging indicated that the foci were not stationary. They fused with or separated from each other (Video S1) and were mobile, sometimes exhibiting long distance movements (Video S2). When immunostained with Golgi marker GM130 in red (Fig. 3A, right panels), the optineurin foci in RGC5 cells did not colocalize with the Golgi apparatus (Fig. 3A, insert, bottom right panel). The lack of major colocalization, confirmed by confocal microscopy after sequential scanning, was also demonstrated previously in human RPE and trabecular meshwork cells [17]. Golgi fragmentation (Fig. 3A, arrow) was in addition noted in all these cell types [17].

Bottom Line: It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein.Treatment of nocadazole resulted in dispersion of the optineurin foci.The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois, United States of America.

ABSTRACT

Background: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.

Methodology/principal findings: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP) fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time.

Conclusions/significance: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.

Show MeSH
Related in: MedlinePlus