Genetic dissection of endocytic trafficking in drosophila using a horseradish peroxidase-Bride of Sevenless chimera

hook is required for normal maturation of multivesicular endosomes

Arisa Sunio, Anne B. Metcalf, Helmut Krämer

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Mutations in the hook gene alter intracellular trafficking of internalized ligands in Drosophila. To dissect this defect in more detail, we developed a new approach to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of HRP fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera was fully functional: it rescued the boss mutant phenotype, and its trafficking was indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss was internalized into R7 cells. In wild-type tissue, Boss accumulated in mature multivesicular bodies (MVBs) within R7 cells; such accumulation was not observed in hook eye disks, however. Quantitative electron microscopy revealed a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes were detected. Our genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes.

Original languageEnglish (US)
Pages (from-to)847-859
Number of pages13
JournalMolecular Biology of the Cell
Volume10
Issue number4
StatePublished - Apr 1999

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Endosomes
Horseradish Peroxidase
Drosophila
Dissection
Multivesicular Bodies
Ligands
Lysosomes
Electron Microscopy
Proteins
Phenotype
Mutation
Genes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

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title = "Genetic dissection of endocytic trafficking in drosophila using a horseradish peroxidase-Bride of Sevenless chimera: hook is required for normal maturation of multivesicular endosomes",
abstract = "Mutations in the hook gene alter intracellular trafficking of internalized ligands in Drosophila. To dissect this defect in more detail, we developed a new approach to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of HRP fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera was fully functional: it rescued the boss mutant phenotype, and its trafficking was indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss was internalized into R7 cells. In wild-type tissue, Boss accumulated in mature multivesicular bodies (MVBs) within R7 cells; such accumulation was not observed in hook eye disks, however. Quantitative electron microscopy revealed a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes were detected. Our genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes.",
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