The Effect Of CDC50A And Rab10 On Phosphatidylserine Flip And Subcellular Localization

Phosphatidylserine(PS),as the most abundant anion phospholipid in eukaryotic cells,is essential for cell apoptosis,blood clotting,vesicle budding,cargo transport,cell adhesion,migration,etc.In mammals,PS synthesis defects will not survive,and over production will lead to congenital lenz-majewski syndrome.Therefore,exploring regulation mechanism of PS localization and flip will help us elucidating the molecular mechanism of various activities in cells and the treatment of diseases.The asymmetrical distribution of PS on the membrane is mainly regulated by P4-ATPase,which can specifically translocate phospholipids from the extracellular(or lumenal)to the cytoplasmic leaflet of the lipid bilayer.ATP8A1 is a member of P4-ATPase family.CDC50 A,as the ? subunit ATP8A1,play significance roles in ATP8A1 maturing from ER and flippase activity.Rab10,as a small G protein,is a key regulator of vesicle transport and generally participates in a wide range of life activities when activated.Our ongoing research shows that constitution-activated Rab10Q68 L can affect the localization of ATP8A1.Although both CDC50 A and Rab10Q68 L have an impact on the localization of ATP8A1,whether CDC50 A and Rab10Q68 L will regulate the flip,localization and function of PS still needs to be further explored.In this study,Hela cells were used as the research carrier,and confocal microscopic imaging technology was used to investigate whether CDC50 A and Rab10Q68 L can affect the localization of P4-ATPase represented by ATP8A1,and then affect the flip and localization of PS and cell physiological functions,such as cell adhesion and spread.Firstly,the transfected expression of GFP-Lact-C2 plasmid and the exogenous incubation of r EGFP-LACT-C2 protein were used to label PS.Both methods showed that PS was mainly located in the plasma membrane and recycling endosome(RE).Subsequently,CDC50 A knockdown by sh RNA resulted in the retention of ATP8A1 in the endoplasmic reticulum.Further studies show that when CDC50 A was knocked down,the fluorescence intensity of m Cherry-Lact-C2 on the cytoplasmic side of PS was reduced,while the fluorescence of Annexin V-FITC on the extracellular side of the plasma membrane was emerged,suggesting that CDC50 A knockdown inhibited the flip of PS on plasma membrane.Meanwhile,it's worth noting that the fluorescence intensity of m Cherry-Lact-C2 was also significantly reduced on RE,suggesting that the knockdown of CDC50 A also inhibited the flip of PS on endosomes.However,knocking down ATP8A1 had no effect on the fluorescence intensity of m Cherry-Lact-C2,suggesting that other redundant PS flippases compensated for the function of ATP8A1.To sum up,we believe that when CDC50 A is down-expressed,it will inhibit the PS-specific flippases represented by ATP8A1 maturing from ER,thus inhibiting the flip of PS on plasma membrane and endosomes.ATP8A1 translocated from the ER to the endosome and plasma membrane upon co-expresion of CDC50 A and display a large increase in plasma membrane abundance.Lact-C2 labeled PS were also more distributed in the plasma membrane,which may be caused by the increased PS level on the cytoplasmic side of the plasma membrane caused by the flippase activity of ATP8A1.Overexpression of Rab10Q68 L redistributes ATP8A1 to perinuclear region,and redistributes PS to Golgi.Further adhesion and spreading experiments found that both CDC50 A knockdown and Rab10Q68 L overexpression inhibited cell adhesion and spreading ability,indicating that the change of PS or subcellular localization may affect cell movement.Our results provide clues for studies on the regulation mechanism of PS flip and localization.