果蠅GOLPH3(Rotini)調控Pipe的逆向運輸並影響背腹軸的發育

Abstract

The dorsal-ventral (D/V) axis of Drosophila embryo is determined by two signaling pathways: the dorsal side of embryo is regulated by EGFR pathway in follicle cells, and ventral side is mediated by the serine protease cascade. Pipe encodes a sulfotransferase that functions in Golgi of follicle cells to activate the serine protease cascade. Finally, the transcription factor, Dorsal, enters the nucleus to determine the development of ventral side of embryo. The pipe mutants show a dorsalized embryo. Rotini (rti), a homologue of human GOLPH3 protein, has been known to be involved in HSPGs modification. In addition, rti germline clone (GLC) shows a dorsalized embryo and affects the distribution of Dorsal, and rti follicle cell clone causes denticle belts disappeared. It suggests both rti in germline and follicle cells can influence the D/V polarity. Previous studies reveal rti interacts with Pipe in Drosophila S2 cells, and Pipe subcellular localization is altered with rti dosage in follicle cells. The thesis is aimed to investigate how rti of follicle cells regulate Pipe trafficking to affect the D/V polarity. The wing imaginal discs of Drosophila are utilized because of controllable genotype, large cell numbers, and its typical Golgi morphology is suitable to study Pipe under electron microscope. The results show Pipe is localized accurately in wing disc cells, and the overlapping area with cis-Golgi marker increases when rti is overexpressed, but decreases when rti is knocked down. It indicates that subcellular distribution of Pipe in disc cells is consistent with follicle cells, and suggests rti may regulate the retrograde trafficking of Pipe in Golgi. Pipe subcellular localization is affected in COPI or COPII mutants, and rti overexpression can suppress Pipe mislocalization in COPI and COPII mutants, representing that Rti is involved in both anterograde and retrograde trafficking. Under electron microscope, Pipe is distributed in the whole Golgi, but accumulated in mid- and cis-Golgi when rti is overexpressed. However, Pipe is distributed in cytosol, multivesicular bodies and lysosomes when rti is knocked down. It is further found Pipe is not accumulated in ER when rti is knocked down, suggesting Rti may be involved in a COPII-independent anterograde pathway. In co-immunoprecipitation assay, Pipe can interact with COPI and COPII, but Rti only interact with COPI. In conclusion, rti can interact with COPI to mediate retrograde trafficking of Pipe. In addition, the retrograde degree is dependent on Rti dosage. Based on these results, rti in follicle cells can interact with COPI to mediate Pipe subcellular localization by retrograde and influence D/V polarity of Drosophila embryo.