Study On The Functional Conservation Of The Intracellular Domain Of The Polar Protein Crumbs

The establishment and maintenance of epithelial apical-basal polarity rely on the proper expression and localization of various polarity proteins.Crumbs(Crb)is a vital protein that is essential for epithelial polarity in Drosophila.It,s maiuly located on the top of cell membrane,and exists as an apical determinant.Crb is a transmembrane protein,consists of a large extracellular part and a small intracellular part.Though the intracellular part only contains 37 amino acides,it plays important functions during the polarity establishment.This intracellular part consists of two functional related domains:FERM-domain binding site(FDB)and PDZ-domain binding site(PDB).These two domains are both indispensable for the restoration of crb mutation phenotype in Drosophila and mammalian cells,however,they participate in different physiological processes by interacting with different proteins.PDB is mainly involved in the combinationwith the PDZ domain of Stardust(Sdt)and dPar6,which is the critical step for the expressing and locating of Crb complex;FDB is mainly combined with cytoskeleton related regulatory proteins.The sequence of the Crb intracellular domain is highly conserved in Drosophila,Caenorhabditis elegans and human,thus we want to investigate whether the function of intracellular domain of Crb is also conserved by genetic methods.The embryonic epithelial cell of the Drosophila is an ideal model to study the mechanism of apical-basal polarity establishment.In this study,we change the intracellular part of the endogenous Crb of Drosophila to that of the CeCrb2 of C elegans by genomic engineering.Moreover,to detect the cellular localization of the proteins,we labelled the chimeric protein Crb-ce2 and the endogenous Crb protein with HA tags respectively.These two new lines are named Crb-ce2::HA and Crb::HA.We found that,unlike the lethal FDB motif and PDB motif mutants.Crb-ce2::HA can survive as homozygous mutant and show similar phenotypes and embryo survival rate to wild type flies.The cellular localization of Crb and other polarity proteins is detected by immunofluorescent staining in embryonic epithelial cells of Crb-ce2::HA and Crb::HA.The results showed that the expression and localization of polarity proteins are normal in Crb-ce2::HA mutant,which indicate that the intracellular domain of CeCrb2 can function properly and fully rescue the polarity defects of FDB and PDB motif mutants in Drosophila.All these results suggest that the function of Crb intracellular part is conserved between Drosophila and C.elegans.In this study,the intracellular part of the endogenous Crb is replaced by that of CeCrb2 by a genetic tool.Phenotype,viability and cell biology analysis showed that the replacement does not affect Crb,s function during the polarity establishment,which shows that the intracellular part of the Crb is conserved not only in sequence,but also in function.