A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells.

Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organizes an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy.

Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6.

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins.