Mechanisms Of Store Operated Calcium Entry Mediated UDP Effect On Microglia Phagocytosis

BackgroundMicroglia are resident macrophages in the central nervous system(CNS)and the main immune defense system of the central nervous system(CNS).Microglia cells can phagocytose cellular debri and other cellular components.Microglia cells have a variety of membrane receptors which can activate intracellular Ca2+signaling.Store operated calcium entry(SOCE)is a in cellular calcium signalling and in maintaining cellular calcium balance,which has been proved to exist in microglia cells.However,little is known about the function of calcium operated calcium entry in microglia cells.Our previous study has shown that uridine diphosphate(UDP)could activate store operated calcium entry,which mediates the UDP effects on microglia phagocytosis.PurposeTherefore,in this experiment,we will study the molecular mechanisms of how UDP induced SOCE can boost microglia phagocytosis.Method1.To study the relationship between P2Y6 receptor and SOCE in microglia:1.1 RT-PCR was used to detect the express of various P2Y receptor subtypes in microglia cells,including P2Y2,P2Y4,P2Y6 and P2Y12;1.2 C8-B4 cells were loaded with Fluo-4 and confocal microscopy was used to measure[Ca2+].P2Y6 receptor selective agonist 5-OMe-UDP was applied to induce SOCE.2.To study the effect of UDP induced SOCE on F-actin and membrane ruffling in microglia:2.1 Confocal microscopy was used to record membrane ruffling and F-actin depolymerizing agent cytochalasin was applied to study if membrane rufling is dependent on F-actin.Fluorescence microscopy of LifeAct-GFP,which binds F-actin and bright field imaging of membrane ruffling were applied to study the relationship between membrane rufing and F-actin;2.2 Application of SOCE inhibitor 2-APB and removal of extracellular Ca2+was used to study if SOCE block could reverse UDP effects on membrane ruffling.3.To study the mechanism of how UDP induced SOCE boost phagocytosis:3.1 Cytochalasin was applied to study if microglia phagocytosis is dependent on F-actin;3.2 Simultaneous imaging of LifeAct-GPF and pHrodo-zymosan particles to observed the dynamics of F-actin during phagocytosis in control and UDP conditions.Result1.UDP can induce SOCE by activating P2Y6 receptor:1.1 C8-B4 cells express P2Y6 receptors;1.2 P2Y6 receptor selective agonist 5-OMe-UDP can induce SOCE.2.UDP induced SOCE can alter the F-actin state of microglia:2.1 Membrane ruffling on C8-B4 cells is dependent on F-actin remodeling;2.2 The effect of UDP on membrane ruffling is mediated by SOCE.3.UDP enhances phagocytosis by affecting F-actin states:3.1 Microglial phagocytosis depends on F-actin remodeling;3.2 SOCE induced by UDP boost phagocytosis by inducing F-actin constriction in phagocytosis cups.ConclusionMicroglia cells express P2Y6 receptors,whose activation could induce SOCE.Calcium flowed into the microglia cells by SOCE could change the F-actin dynamics and thus membrane ruffling.It could also suppress the unproductive ruffling and boost the F-actin constriction in phagocytosis cup during microglia phagocytosis.