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Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

Rodriguez-Fernandez IA, Dell'Angelica EC - PLoS ONE (2015)

Bottom Line: The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family.Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively.These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

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Related in: MedlinePlus

Strategy for the genetic modifier screening carried out in this study.The primary screening involved parental (P0) crosses between male flies carrying a deficiency (Df) over a balancer in an autosomal chromosome (Chr. 2, 3 or 4) and female flies homozygous for the hypomorphic g2 allele of the X-linked gene encoding the δ subunit of AP-3. The eye color of male progeny (F1) carrying one copy of each deficiency (without the balancer) was compared with that of control g2 males and, if deemed different, the corresponding deficiency was selected for a secondary screening involving the same P0 cross followed by quantification of red and brown pigments in the F1 males carrying the deficiency. In cases in which differences in both red and brown pigment content were statistically significant, further validation and fine mapping was attempted using independent deficiency lines in which the deleted genomic regions partially overlapped with that of the deficiency identified through screening. When successful, theses steps allowed identification of a relatively small genomic region (rectangle) containing a modifier gene of interest (red arrow).
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pone.0143026.g001: Strategy for the genetic modifier screening carried out in this study.The primary screening involved parental (P0) crosses between male flies carrying a deficiency (Df) over a balancer in an autosomal chromosome (Chr. 2, 3 or 4) and female flies homozygous for the hypomorphic g2 allele of the X-linked gene encoding the δ subunit of AP-3. The eye color of male progeny (F1) carrying one copy of each deficiency (without the balancer) was compared with that of control g2 males and, if deemed different, the corresponding deficiency was selected for a secondary screening involving the same P0 cross followed by quantification of red and brown pigments in the F1 males carrying the deficiency. In cases in which differences in both red and brown pigment content were statistically significant, further validation and fine mapping was attempted using independent deficiency lines in which the deleted genomic regions partially overlapped with that of the deficiency identified through screening. When successful, theses steps allowed identification of a relatively small genomic region (rectangle) containing a modifier gene of interest (red arrow).

Mentions: In order to identify regions within the autosomal chromosomes of D. melanogaster (chromosomes 2, 3 and 4) potentially bearing genes that, when in hemizygous form due to deletion of one copy, modify the severity of the phenotype of AP-3-mutant flies, a genetic screening was undertaken following the strategy depicted in Fig 1. In both primary (qualitative) and secondary (quantitative) steps of the screening, male flies carrying large multi-gene deletions (‘deficiencies’) from the so-called ‘Bloomington Deficiency Kit’ collections were crossed with female flies homozygous for the g2 mutation (on chromosome X) to obtain F1 males that were hemizygous for g2 and heterozygous for a given deficiency over a normal chromosome; for each deficiency, the eye color of these F1 males was compared to that of control g2 males first by light microscopy and, if selected for secondary screening, by pigment extraction and quantification (Fig 1). The g2 mutation in the gene encoding the δ subunit of AP-3 was chosen not only because of its genomic location on the X chromosome (thus minimizing the number of genetic crosses required to test each deficiency) but also because it represents a weak hypomorph of the garnet allelic series [54]. In our hands, the eyes of male adult flies carrying the g2 allele in the Canton-S genetic background contain 25–30% of the red pigment content of those of the control Canton-S line, whereas those of males carrying one of the strongest alleles of the same gene, g53d, contain less than 5% of wild-type levels of red pigments (see, for example, Ref. [39]). Thus, the g2 line was used as a sensitized strain with which both phenotypic enhancers and suppressors could potentially be identified.


Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

Rodriguez-Fernandez IA, Dell'Angelica EC - PLoS ONE (2015)

Strategy for the genetic modifier screening carried out in this study.The primary screening involved parental (P0) crosses between male flies carrying a deficiency (Df) over a balancer in an autosomal chromosome (Chr. 2, 3 or 4) and female flies homozygous for the hypomorphic g2 allele of the X-linked gene encoding the δ subunit of AP-3. The eye color of male progeny (F1) carrying one copy of each deficiency (without the balancer) was compared with that of control g2 males and, if deemed different, the corresponding deficiency was selected for a secondary screening involving the same P0 cross followed by quantification of red and brown pigments in the F1 males carrying the deficiency. In cases in which differences in both red and brown pigment content were statistically significant, further validation and fine mapping was attempted using independent deficiency lines in which the deleted genomic regions partially overlapped with that of the deficiency identified through screening. When successful, theses steps allowed identification of a relatively small genomic region (rectangle) containing a modifier gene of interest (red arrow).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643998&req=5

pone.0143026.g001: Strategy for the genetic modifier screening carried out in this study.The primary screening involved parental (P0) crosses between male flies carrying a deficiency (Df) over a balancer in an autosomal chromosome (Chr. 2, 3 or 4) and female flies homozygous for the hypomorphic g2 allele of the X-linked gene encoding the δ subunit of AP-3. The eye color of male progeny (F1) carrying one copy of each deficiency (without the balancer) was compared with that of control g2 males and, if deemed different, the corresponding deficiency was selected for a secondary screening involving the same P0 cross followed by quantification of red and brown pigments in the F1 males carrying the deficiency. In cases in which differences in both red and brown pigment content were statistically significant, further validation and fine mapping was attempted using independent deficiency lines in which the deleted genomic regions partially overlapped with that of the deficiency identified through screening. When successful, theses steps allowed identification of a relatively small genomic region (rectangle) containing a modifier gene of interest (red arrow).
Mentions: In order to identify regions within the autosomal chromosomes of D. melanogaster (chromosomes 2, 3 and 4) potentially bearing genes that, when in hemizygous form due to deletion of one copy, modify the severity of the phenotype of AP-3-mutant flies, a genetic screening was undertaken following the strategy depicted in Fig 1. In both primary (qualitative) and secondary (quantitative) steps of the screening, male flies carrying large multi-gene deletions (‘deficiencies’) from the so-called ‘Bloomington Deficiency Kit’ collections were crossed with female flies homozygous for the g2 mutation (on chromosome X) to obtain F1 males that were hemizygous for g2 and heterozygous for a given deficiency over a normal chromosome; for each deficiency, the eye color of these F1 males was compared to that of control g2 males first by light microscopy and, if selected for secondary screening, by pigment extraction and quantification (Fig 1). The g2 mutation in the gene encoding the δ subunit of AP-3 was chosen not only because of its genomic location on the X chromosome (thus minimizing the number of genetic crosses required to test each deficiency) but also because it represents a weak hypomorph of the garnet allelic series [54]. In our hands, the eyes of male adult flies carrying the g2 allele in the Canton-S genetic background contain 25–30% of the red pigment content of those of the control Canton-S line, whereas those of males carrying one of the strongest alleles of the same gene, g53d, contain less than 5% of wild-type levels of red pigments (see, for example, Ref. [39]). Thus, the g2 line was used as a sensitized strain with which both phenotypic enhancers and suppressors could potentially be identified.

Bottom Line: The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family.Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively.These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Show MeSH
Related in: MedlinePlus