Cells continuously take up nutrients and signaling molecules. Internalized proteins destined for degradation are routed from the plasma membrane through early endosomes, multivesicular bodies (MVBs), late endosomes and finally to lysosomes. The molecular mechanisms regulating these steps in endocytic trafficking are important, because they are altered by many genetic diseases including Chediak-Fligashi or Hermansky-Pudlak syndromes. The Drosophila compound eye is an excellent model system for a genetic analysis of endocytic trafficking. Mutations in many genes necessary for this process can be identified as eye color mutations because they also interfere with the delivery of biosynthetic cargo to pigment granules. Internalization of the Boss ligand into R7 photoreceptor cells provides a direct assay to follow endocytic trafficking. This proposal is aimed at exploiting these features of the Drosophila eye for a genetic dissection of endocytic trafficking in multicellular organisms. The (dor) deep orange and (car) carnation eye color genes encode two subunits of a complex that is necessary late in lysosomal delivery and eye pigmentation.
In Specific Aim 1, the rote of additional subunits of this complex in lysosomal delivery will be characterized.
In Specific Aim 2, the specific defects in endocytic and biosynthetic trafficking in dor mutant cells will be determined on the ultrastructural level by labeling different compartments with HRP fusion proteins. To exploit the exciting connection between lysosomal delivery and eye color mutations, Specific Aim 3 proposes a systematic screen for mutants affecting eye color and endocytic trafficking. Besides additional dor-like mutations acting late in the pathway, this screen will also yield mutations interfering with earlier steps in endocytic trafficking, for example, the regulation of MVB biogenesis and function.
Specific Aim 4 proposes the characterization of DmVps28 as an example for such mutations acting early in the endocytic pathway. An important long-term goal of this work is to relate defects in different trafficking mutations to the symptoms in genetic diseases altering endocytic trafficking. To directly compare phenotypes in the Drosophila model system, Specific Aim 5 is directed towards the isolation of mutations in the Drosophila Hermansky-Pudlak syndrome-1 gene and the characterization of the resulting defects in endocytic trafficking.
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