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The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

Milligan SC, Alb JG, Elagina RB, Bankaitis VA, Hyde DR - J. Cell Biol. (1997)

Bottom Line: Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo.Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism.This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

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Histology of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. White-eyed versions (cn bw) of  wild-type (A), rdgB2 (B), rdgB+; P[rdgB-T59E] (C), and rdgB+;  P[pitpα-rdgB] (D) flies were raised in a 12-h light/dark cycle for  <6 d after eclosion. The rdgB2 retinal sections exhibited the reduction and loss of rhabdomeres, formation of holes (long arrows), and condensation of the photoreceptor cell bodies (black  arrowheads). The rdgB+; P[rdgB-T59E] flies revealed a reduction  in the size of the R1-6 outer rhabdomeres, with few holes appearing in the retina. The R1-6 cell bodies appear to be nearly the  same in both the young and old retinas, which is roughly similar  to the wild-type cell bodies. The rdgB+; P[pitpα-rdgB] flies  showed signs of degeneration that more closely matched the  rdgB2 mutant flies, with holes (long arrows) appearing in the retinal sections. Additionally, the microvillar rhabdomeres are beginning to unpack (white arrowheads). Bar, 10 μm.
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Figure 6: Histology of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. White-eyed versions (cn bw) of wild-type (A), rdgB2 (B), rdgB+; P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] (D) flies were raised in a 12-h light/dark cycle for <6 d after eclosion. The rdgB2 retinal sections exhibited the reduction and loss of rhabdomeres, formation of holes (long arrows), and condensation of the photoreceptor cell bodies (black arrowheads). The rdgB+; P[rdgB-T59E] flies revealed a reduction in the size of the R1-6 outer rhabdomeres, with few holes appearing in the retina. The R1-6 cell bodies appear to be nearly the same in both the young and old retinas, which is roughly similar to the wild-type cell bodies. The rdgB+; P[pitpα-rdgB] flies showed signs of degeneration that more closely matched the rdgB2 mutant flies, with holes (long arrows) appearing in the retinal sections. Additionally, the microvillar rhabdomeres are beginning to unpack (white arrowheads). Bar, 10 μm.

Mentions: We compared the histology of the dominantly degenerating retinas to each other and with rdgB2 to determine if they could be undergoing the same process. We sectioned retinas from 6-d-old white-eyed (cn bw) rdgB+ flies that either lacked or contained P[rdgB-T59E] or P[pitpα-rdgB] (Fig. 6). The 6-d-old rdgB2; cn bw mutant flies (Fig. 6 B) exhibited the characteristic degeneration phenotype of rhabdomere loss, perforations of the retina, and the appearance of optically dense photoreceptor cell bodies. The rdgB+; P[rdgB-T59E] retinas exhibited significantly fewer retinal perforations and dense photoreceptor cell bodies relative to rdgB2 flies (Fig. 6 C). Most strikingly, the rdgB+; P[rdgB-T59E] retinas lacked mature R1-6 rhabdomeres (Fig. 6 C). Indeed, the rhabdomeres of newly eclosed rdgB+; P[rdgB-T59E] flies were less well developed compared to wild-type controls, and diminished in size as the flies aged (data not shown). This rhabdomere atrophy of photoreceptors R1-6 resembled the hypomorphic ninaE mutant phenotype, which results from a significant reduction in rhodopsin expression in photoreceptors R1-6 (Leonard et al., 1992; Kumar and Ready, 1995). The dominant pitpα-rdgB degeneration morphology was more similar to the rdgB2 phenotype, with the most striking defects being the numerous perforations in the retina and the reduction in R1-6 rhabdomere size relative to R7 (Fig. 6 D). Additionally, the R1-6 microvillar rhabdomeres began to exhibit signs of unpacking (Fig. 6 D) that we had not previously observed in any rdgB mutants. Thus, while the dominant rdgB-T59E mutant phenotype approximated the ninaE hypomorphic phenotype, the dominant pitpα-rdgB phenotype was morphologically more like the rdgB mutant retina with some additional mutant characteristics.


The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

Milligan SC, Alb JG, Elagina RB, Bankaitis VA, Hyde DR - J. Cell Biol. (1997)

Histology of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. White-eyed versions (cn bw) of  wild-type (A), rdgB2 (B), rdgB+; P[rdgB-T59E] (C), and rdgB+;  P[pitpα-rdgB] (D) flies were raised in a 12-h light/dark cycle for  <6 d after eclosion. The rdgB2 retinal sections exhibited the reduction and loss of rhabdomeres, formation of holes (long arrows), and condensation of the photoreceptor cell bodies (black  arrowheads). The rdgB+; P[rdgB-T59E] flies revealed a reduction  in the size of the R1-6 outer rhabdomeres, with few holes appearing in the retina. The R1-6 cell bodies appear to be nearly the  same in both the young and old retinas, which is roughly similar  to the wild-type cell bodies. The rdgB+; P[pitpα-rdgB] flies  showed signs of degeneration that more closely matched the  rdgB2 mutant flies, with holes (long arrows) appearing in the retinal sections. Additionally, the microvillar rhabdomeres are beginning to unpack (white arrowheads). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Histology of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. White-eyed versions (cn bw) of wild-type (A), rdgB2 (B), rdgB+; P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] (D) flies were raised in a 12-h light/dark cycle for <6 d after eclosion. The rdgB2 retinal sections exhibited the reduction and loss of rhabdomeres, formation of holes (long arrows), and condensation of the photoreceptor cell bodies (black arrowheads). The rdgB+; P[rdgB-T59E] flies revealed a reduction in the size of the R1-6 outer rhabdomeres, with few holes appearing in the retina. The R1-6 cell bodies appear to be nearly the same in both the young and old retinas, which is roughly similar to the wild-type cell bodies. The rdgB+; P[pitpα-rdgB] flies showed signs of degeneration that more closely matched the rdgB2 mutant flies, with holes (long arrows) appearing in the retinal sections. Additionally, the microvillar rhabdomeres are beginning to unpack (white arrowheads). Bar, 10 μm.
Mentions: We compared the histology of the dominantly degenerating retinas to each other and with rdgB2 to determine if they could be undergoing the same process. We sectioned retinas from 6-d-old white-eyed (cn bw) rdgB+ flies that either lacked or contained P[rdgB-T59E] or P[pitpα-rdgB] (Fig. 6). The 6-d-old rdgB2; cn bw mutant flies (Fig. 6 B) exhibited the characteristic degeneration phenotype of rhabdomere loss, perforations of the retina, and the appearance of optically dense photoreceptor cell bodies. The rdgB+; P[rdgB-T59E] retinas exhibited significantly fewer retinal perforations and dense photoreceptor cell bodies relative to rdgB2 flies (Fig. 6 C). Most strikingly, the rdgB+; P[rdgB-T59E] retinas lacked mature R1-6 rhabdomeres (Fig. 6 C). Indeed, the rhabdomeres of newly eclosed rdgB+; P[rdgB-T59E] flies were less well developed compared to wild-type controls, and diminished in size as the flies aged (data not shown). This rhabdomere atrophy of photoreceptors R1-6 resembled the hypomorphic ninaE mutant phenotype, which results from a significant reduction in rhodopsin expression in photoreceptors R1-6 (Leonard et al., 1992; Kumar and Ready, 1995). The dominant pitpα-rdgB degeneration morphology was more similar to the rdgB2 phenotype, with the most striking defects being the numerous perforations in the retina and the reduction in R1-6 rhabdomere size relative to R7 (Fig. 6 D). Additionally, the R1-6 microvillar rhabdomeres began to exhibit signs of unpacking (Fig. 6 D) that we had not previously observed in any rdgB mutants. Thus, while the dominant rdgB-T59E mutant phenotype approximated the ninaE hypomorphic phenotype, the dominant pitpα-rdgB phenotype was morphologically more like the rdgB mutant retina with some additional mutant characteristics.

Bottom Line: Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo.Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism.This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

Show MeSH
Related in: MedlinePlus