Untersuchungen zu molekularen und genetischen Ursachen Licht-abhängiger retinaler Degenration bei Drosophila melanogaster
Final Report Abstract
Photoreceptor cells require highly elaborated mechanisms to ensure cell survival despite tremendous light stress, and defects in these mechanisms lead to photoreceptor degeneration and blindness. In human about 50 genes have been associated with blindness. Many of these genes, which are involved in photoreceptor development and homeostasis, are structurally and functionally conserved in Drosophila and similarly lead to retinal degeneration when mutated. This makes the fly eye an ideal system to study the cellular and molecular basis of blindness, despite the fact that vertebrate and fly eyes are differently organised. Our research concentrates on the evolutionarily conserved Crumbs (Crb) protein complex. Mutations in any core member of the Crumbs (Crb) complex (i. e. the transmembrane protein Crb, the membrane-associated guanylate kinase protein Stardust (Sdt) and the PDZ domain– containing proteins DPATJ and DLin7) lead to retinal degeneration in Drosophila. Strikingly, mutations in the human orthologue, CRB1, result in Retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA), two severe forms of retinal dystrophies leading to early onset blindness. Crb is a large transmembrane protein, which was first identified as an apical determinant in Drosophila melanogaster embryonic epithelia, where it is required for the maintenance of apico-basal polarity. Given the complexity in function of the Crb complex and the complex organization of both Crb and Sdt, we were interested to find out whether individual isoforms and/or individual protein domains have specific functions during development and homeostasis of photoreceptor cells. Therefore, we performed a structure-function analysis of Crb. We could show that the full length protein and the membrane-bound intracellular domain of Crb can rescue all morphogenetic defects observed in crb mutant photoreceptor cells, while the extracellular domain can only restore some aspects. Overexpression of the membrane-bound intracellular domain of Crb can induce a second apical pole in photoreceptor cells when expressed early during pupal development, indicating that Crb can act as an “apical determinant”. Our analysis further showed that sdt is a complex genetic locus, in that different isoforms are expressed at different developmental stages and perform different functions. For example, two of the isoforms expressed in photoreceptor cells have antagonistic functions with respect to retinal degeneration, in that the expression of one isoform promotes retinal degeneration, while the other prevents it. Taken together, our work has contributed to unraveling the molecular functions of two key members of the Crb complex, which will contribute to better understand the function of this highly conserved protein complex in other contexts and in other organisms.
Publications
- (2007) Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration. Genetics. 176, 2189-2200
Berger S, B. N., Grawe F, Johnson K, Knust E
- (2008) Multiple domains of Drosophila Stardust differentially mediate localisation of the Crumbs/Stardust complex during photoreceptor development. J Cell Sci 121, 2018-2026
Bulgakova, N. A., Kempkens, Ö., and Knust, E.
- (2009) A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells. Eur J Cell Biol. 88, 765-777
Richard, M., Muschalik, N., Grawe, F., Ozüyaman, S., and Knust E.
- (2009) The Crumbs complex. J Cell Sci 122, 2587-2596
Bulgakova, N. A., and Knust, E.
- (2010) Antagonistic Functions of Two Stardust Isoforms in Drosophila Photoreceptor Cells. Mol Biol Cell 21, 3915-3925
Bulgakova, N. A., Rentsch, M., and Knust, E.
- (2011) Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors. J Cell Sci 24, 1–11
Muschalik, N., and Knust, E.
