A Fluorescence Spectroscopic Approach To Quantify The Binding Characteristics Of The Intracellular Domain Of Crumbs With Pdz Domain-containing Proteins In Drosophila Melanogaster

Abstract

Formation of multiprotein complexes is a common strategy to pattern a cell, thereby generating spatially and functionally distinct entities at specialized 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 and/or the developmental status. The large transmembrane protein Crumbs is required for the establishment and maintenance of apico-basal polarity in Drosophila embryonic epithelia. The short intracellular domain of Crumbs localizes an evolutionary conserved protein scaffold via its interaction with the single PDZ-domain of Stardust. The Crumbs/Stardust/DPATJ complex coexists in Drosophila epithelial cells with another apical protein complex consisting of Bazooka, DmPar-6 and DaPKC. The degree of spatial overlap between components of these two complexes found in the subapical regions of many epithelia is striking and several in silico, qualitative in vitro and in vivo interaction experiments performed with constituents of these complexes have all pointed to a direct interaction between Crumbs and DmPar-6. To investigate and compare the interaction of the intracellular domain of Crumbs with the PDZ domains of Stardust and DmPar-6 we labeled the (putative) interaction partners with fluorescent dyes. This enables us to quantify the respective binding characteristics and complex properties with single molecule and ensemble FRET- and anisotropy- as well as with stopped flow-measurements.