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The protein O-glucosyltransferase Rumi modifies eyes shut to promote rhabdomere separation in Drosophila.

Haltom AR, Lee TV, Harvey BM, Leonardi J, Chen YJ, Hong Y, Haltiwanger RS, Jafar-Nejad H - PLoS Genet. (2014)

Bottom Line: In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells.The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys.Therefore, the role of O-glucose in regulating Eys may be conserved.

View Article: PubMed Central - PubMed

Affiliation: Program in Genes & Development, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, United States of America; Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.

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Related in: MedlinePlus

Loss of the enzymatic function of Rumi results in rhabdomere adhesion.Shown are electron micrographs of a single ommatidium from adult (A–E,H,I) or 65% PD (J,K) from the indicated genotypes. All animals were raised at 18°C. (A) Wild-type. Arrows indicate rhabdomeres and asterisks indicate the IRS. Scale bar: 2 µm. (B) 1-day old rumi−/−. Note the attachment in the neighboring rhabdomeres. (C) 1-day old rumi−/− expressing a wild-type P{rumigt-FLAG} genomic transgene. (D,E) The rumi−/− rhabdomere attachment phenotype does not change with age, since 15-day old (D) and 40-day old (E) animals show a similar degree of attachment. (F) Percentage of number of individual rhabdomeres per ommatidia for various genotypes. At least three animals were used for each genotype. The number of ommatidia examined for each genotype is as follows: wt (50), 1d (35), 15d (66), 40d (85), rescue (126). (G) Quantification of average individual rhabdomere number per ommatidium for the data shown in F. Rhabdomere attachments in 1-day, 15-day and 40-day old rumi animals are not significantly different from one another, but are significantly different from wild-type and rescued animals (*P<0.0001). NS, not significant. (H,I) The enzymatically inactive allele rumi79 also shows a rhabdomere attachment phenotype (H), which can be rescued by one copy of the wild-type P{rumigt-FLAG} genomic transgene (I). (J,K) Ommatidia from animals at 65% PD from wild-type (J) and rumi−/− (K). Arrowheads in (K) indicate points of rhabdomere attachment. (L) Means ± SEM of IRS area in wild-type and rumi−/− at 65% PD. IRS areas were measured using ImageJ software. Unpaired t-test was used to compare wt (n = 14) and rumi−/− (n = 24) IRS, *P<0.0001.
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pgen-1004795-g001: Loss of the enzymatic function of Rumi results in rhabdomere adhesion.Shown are electron micrographs of a single ommatidium from adult (A–E,H,I) or 65% PD (J,K) from the indicated genotypes. All animals were raised at 18°C. (A) Wild-type. Arrows indicate rhabdomeres and asterisks indicate the IRS. Scale bar: 2 µm. (B) 1-day old rumi−/−. Note the attachment in the neighboring rhabdomeres. (C) 1-day old rumi−/− expressing a wild-type P{rumigt-FLAG} genomic transgene. (D,E) The rumi−/− rhabdomere attachment phenotype does not change with age, since 15-day old (D) and 40-day old (E) animals show a similar degree of attachment. (F) Percentage of number of individual rhabdomeres per ommatidia for various genotypes. At least three animals were used for each genotype. The number of ommatidia examined for each genotype is as follows: wt (50), 1d (35), 15d (66), 40d (85), rescue (126). (G) Quantification of average individual rhabdomere number per ommatidium for the data shown in F. Rhabdomere attachments in 1-day, 15-day and 40-day old rumi animals are not significantly different from one another, but are significantly different from wild-type and rescued animals (*P<0.0001). NS, not significant. (H,I) The enzymatically inactive allele rumi79 also shows a rhabdomere attachment phenotype (H), which can be rescued by one copy of the wild-type P{rumigt-FLAG} genomic transgene (I). (J,K) Ommatidia from animals at 65% PD from wild-type (J) and rumi−/− (K). Arrowheads in (K) indicate points of rhabdomere attachment. (L) Means ± SEM of IRS area in wild-type and rumi−/− at 65% PD. IRS areas were measured using ImageJ software. Unpaired t-test was used to compare wt (n = 14) and rumi−/− (n = 24) IRS, *P<0.0001.

Mentions: When raised at 18°C, rumi mutant animals are viable and show only a mild loss of Notch signaling in some contexts [12], [14]. To explore whether Rumi plays a role in rhabdomere morphogenesis and IRS formation, we raised animals homozygous for the protein- allele rumiΔ26 (rumi−) in ambient light at 18°C and performed transmission electron microscopy (TEM) on adult fly eyes. In cross sections of wild-type retinas, the rhabdomeres of the seven visible photoreceptor cells are separated from neighboring rhabdomeres by the IRS [19] (Figure 1A and 1F). However, 1-day old rumi−/− animals exhibited a moderate, yet 100% penetrant, rhabdomere attachment phenotype, i.e. attachment of two or more rhabdomeres per ommatidium (Figure 1B and 1F). This phenotype can be fully rescued by P{rumigt-FLAG} (Figure 1C and 1F), a genomic transgene expressing a FLAG-tagged version of Rumi [12], indicating that attachment of rhabdomeres observed in rumi−/− flies is due to the loss of rumi. Sections of rumi−/− animals at 15 and 40 days of age show a similar degree of rhabdomere attachment, suggesting that the phenotype is not age-dependent (Figure 1D–G). Together, these observations indicate that Rumi is required for optical isolation of individual photoreceptors in the Drosophila eye.


The protein O-glucosyltransferase Rumi modifies eyes shut to promote rhabdomere separation in Drosophila.

Haltom AR, Lee TV, Harvey BM, Leonardi J, Chen YJ, Hong Y, Haltiwanger RS, Jafar-Nejad H - PLoS Genet. (2014)

Loss of the enzymatic function of Rumi results in rhabdomere adhesion.Shown are electron micrographs of a single ommatidium from adult (A–E,H,I) or 65% PD (J,K) from the indicated genotypes. All animals were raised at 18°C. (A) Wild-type. Arrows indicate rhabdomeres and asterisks indicate the IRS. Scale bar: 2 µm. (B) 1-day old rumi−/−. Note the attachment in the neighboring rhabdomeres. (C) 1-day old rumi−/− expressing a wild-type P{rumigt-FLAG} genomic transgene. (D,E) The rumi−/− rhabdomere attachment phenotype does not change with age, since 15-day old (D) and 40-day old (E) animals show a similar degree of attachment. (F) Percentage of number of individual rhabdomeres per ommatidia for various genotypes. At least three animals were used for each genotype. The number of ommatidia examined for each genotype is as follows: wt (50), 1d (35), 15d (66), 40d (85), rescue (126). (G) Quantification of average individual rhabdomere number per ommatidium for the data shown in F. Rhabdomere attachments in 1-day, 15-day and 40-day old rumi animals are not significantly different from one another, but are significantly different from wild-type and rescued animals (*P<0.0001). NS, not significant. (H,I) The enzymatically inactive allele rumi79 also shows a rhabdomere attachment phenotype (H), which can be rescued by one copy of the wild-type P{rumigt-FLAG} genomic transgene (I). (J,K) Ommatidia from animals at 65% PD from wild-type (J) and rumi−/− (K). Arrowheads in (K) indicate points of rhabdomere attachment. (L) Means ± SEM of IRS area in wild-type and rumi−/− at 65% PD. IRS areas were measured using ImageJ software. Unpaired t-test was used to compare wt (n = 14) and rumi−/− (n = 24) IRS, *P<0.0001.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4238978&req=5

pgen-1004795-g001: Loss of the enzymatic function of Rumi results in rhabdomere adhesion.Shown are electron micrographs of a single ommatidium from adult (A–E,H,I) or 65% PD (J,K) from the indicated genotypes. All animals were raised at 18°C. (A) Wild-type. Arrows indicate rhabdomeres and asterisks indicate the IRS. Scale bar: 2 µm. (B) 1-day old rumi−/−. Note the attachment in the neighboring rhabdomeres. (C) 1-day old rumi−/− expressing a wild-type P{rumigt-FLAG} genomic transgene. (D,E) The rumi−/− rhabdomere attachment phenotype does not change with age, since 15-day old (D) and 40-day old (E) animals show a similar degree of attachment. (F) Percentage of number of individual rhabdomeres per ommatidia for various genotypes. At least three animals were used for each genotype. The number of ommatidia examined for each genotype is as follows: wt (50), 1d (35), 15d (66), 40d (85), rescue (126). (G) Quantification of average individual rhabdomere number per ommatidium for the data shown in F. Rhabdomere attachments in 1-day, 15-day and 40-day old rumi animals are not significantly different from one another, but are significantly different from wild-type and rescued animals (*P<0.0001). NS, not significant. (H,I) The enzymatically inactive allele rumi79 also shows a rhabdomere attachment phenotype (H), which can be rescued by one copy of the wild-type P{rumigt-FLAG} genomic transgene (I). (J,K) Ommatidia from animals at 65% PD from wild-type (J) and rumi−/− (K). Arrowheads in (K) indicate points of rhabdomere attachment. (L) Means ± SEM of IRS area in wild-type and rumi−/− at 65% PD. IRS areas were measured using ImageJ software. Unpaired t-test was used to compare wt (n = 14) and rumi−/− (n = 24) IRS, *P<0.0001.
Mentions: When raised at 18°C, rumi mutant animals are viable and show only a mild loss of Notch signaling in some contexts [12], [14]. To explore whether Rumi plays a role in rhabdomere morphogenesis and IRS formation, we raised animals homozygous for the protein- allele rumiΔ26 (rumi−) in ambient light at 18°C and performed transmission electron microscopy (TEM) on adult fly eyes. In cross sections of wild-type retinas, the rhabdomeres of the seven visible photoreceptor cells are separated from neighboring rhabdomeres by the IRS [19] (Figure 1A and 1F). However, 1-day old rumi−/− animals exhibited a moderate, yet 100% penetrant, rhabdomere attachment phenotype, i.e. attachment of two or more rhabdomeres per ommatidium (Figure 1B and 1F). This phenotype can be fully rescued by P{rumigt-FLAG} (Figure 1C and 1F), a genomic transgene expressing a FLAG-tagged version of Rumi [12], indicating that attachment of rhabdomeres observed in rumi−/− flies is due to the loss of rumi. Sections of rumi−/− animals at 15 and 40 days of age show a similar degree of rhabdomere attachment, suggesting that the phenotype is not age-dependent (Figure 1D–G). Together, these observations indicate that Rumi is required for optical isolation of individual photoreceptors in the Drosophila eye.

Bottom Line: In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells.The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys.Therefore, the role of O-glucose in regulating Eys may be conserved.

View Article: PubMed Central - PubMed

Affiliation: Program in Genes & Development, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, United States of America; Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.

Show MeSH
Related in: MedlinePlus