To Explore The Distinct Membrane Reforming Activities Induced By The Domains Of Abba1 In COS7 Cells

Background & Objective: The IMD domain family plays a central role in membrane protrusion. Unlike MIM and IRSp53, which induce microspikes and dendritic respectively, overexpression of Abba1 induces the formation of membrane ruffling. Interestingly, the cells overexpressing Abba1's IMD alone failed to mediate membrane ruffling, instead of microspikes. The resarch is to explore the distinct membrane reforming activities induced by the domains of Abba1.Methods: Prepared a series of plasmids, such as pEGFP, pEGFP-Abba1, pEGFP-Abba1-IMD(4178),pEGFP-hAbba1-del-WH2(4177),pEGFP-hAbba1-del-SRD(4210),pEGFP-hIRSp53-IMD-hAbba1-SRD1-SRD2-WH2(4764),pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like(4842),pEGFP-hAbba1-IMD-(Lys13>Glu13)-SRD1-SRD2-WH2(5160),pEGFP-hAbba1-IMD-SRD1-SRD2-(Ser>Ala)-WH2(5244),pEGFP-hAbba1-del-357-553aa-WH2(5231),pEGFP-hAbba1-del-554-722aa-WH2(5399),pEGFP-Abba1-del-357-722aa-WH2(5232). COS7 cells were transiently transfected with these plasmids, respectively. Immunofluorescent microscopy was carried out to detect the formation of membrane protrusions. And GTP-bound Rac1(GTP-Rac1) in cell lysates was determined by pull-down with GST-PAK-CRIB followed by Western blot with Rac1 antibody. Results: According to the results of genetic sequencing and double restriction endonuclease map, the recombined plasmids were identified. From immunofluorescent microscopy, unlike overexpression of Abba1,4764(pEGFP-hIR SP53-IMD-hAbba1-SRD1-SRD2-WH2),4177(pEGFP-hAbba1-del-WH2),4210(pEGFP-hAbba1-del-SRD),5244(pEGFP-hAbba1-IMD-SRD1-SRD2-(Ser>Ala)-WH2), 5231(pEGFP-hAbba1-del-357-553aa-WH2),5399(pEGFP-hAbba1-del-554-722aa-WH2), which induce the formation of membrane ruffling, cells overexpressing 5160(pEGFP-hAbba1-IMD-(Lys13>Glu13)-SRD1-SRD2-WH2) could not play this role. Furthermore, COS7 cells overexpressing 4178(pEGFP-hAbba1-IMD) , 4842(pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like),5232(pEGFP-hAbba1-del-357-722aa-WH2)displayed microspikes. Pull-down combined Western blot analysis revealed that the relative level of GTP-Rac1 was raised during overexpression of Abba1. However, the reverse level was measured when transfecting with 4178(pEGFP-hAbba1-IMD) and 5232(pEGFP-hAbba1-del-357-722aa-WH2).Conclusion: The data indicated that IMD is the most important domain for full-length Abba1 inducing membrane ruffling. Neither SRD nor WH2, the rest region, 357-722aa sequence, could participated in inducing membrane ruffling in COS7 cells, which was connected with the decrescent level of GTP-Rac1.