| Background The experimental data documented the important role of endogenous electric fields in morphogenesis,embryonic development,and wound healing.The directed current electric fields(DC EFs)may function as a direction cue to guide cell migration,which is called electrotaxis.Many studies showed that the electrotaxis shared similar pathway with chemotaxis.The caffeine was proved to be an inhibitor in chemotaxis of Dictyostelium discoideum by suppressing the synthesis of c AMP,while the effect of caffeine in electrotaxis is unclear.In this study,the role of caffeine in electric fields guiding cell migration is studied.The mechanism underlie is explored in both physiological and molecular levels by various methods.Our study will provide experimental documents in exploring the mechanism of electrotaxis.Methods 1)Exploring the effect of caffeine in electric fields guiding cell migration by microscopy with time lapes image system;2)Exploring the influence of EFs and caffeine on the dynamics of calcium in vivo by confocal microscope;3)Exploring the key genes,such as G protein,PI3 K,PKB,Erk1/2 in electrotaxis and the role of those genes involved in caffeine promoted electrotaxis by using mutant strains combined with Western Bloting.Results 1)Caffeine may promote EF guiding cell migration of Dictyostelium discoideum cells;2)The caffeine promote cell migration speed instantly,while it showed delayed effects on promoting direction of cell migration;3)Intracellular calcium play important roles in EF guiding cell migation.Caffeine promote the intracellular calcium level.Inhibition of calcium flux abolished the promoting effect of caffeine on EF guiding cell migraition;4)G protein(Gα2,Gβ,Gγ,Gα2Gβ,Gα2Gγ),Erk1/2,ras C,PKBA,Pia A and PI3K(1-5)are important proteins involved in the EF guiding cell migration,the deletion of the above genes inhibits the EF guiding cell migration;while the deletion of pi3k(1-2)gene may promote the directness of the EF guiding cell migration;5)Caffeine treatment increased the phosphorylation level of Erk1/2 in the electric field;the deletion of the Gγ subunit inhibited the activation of Erk1/2 phosphorylation by caffeine in the electric field;6)The deletion of ras C,pia A and pi3k(1-5)genes attenuated the promotion effects of caffeine on EF guiding cell migration.Conclusions Unlike chemotaxis,caffeine obviously promotes the EF guiding cell migration of Dictyostelium discoideumby increaseing intracellular calcium.The abolish of calcium flux by inhibitors suppressed the promoting effects of caffeine on EF guiding cell migration.In addition,G protein(Gα2,Gβ,Gγ,Gα2Gβ,Gα2Gγ),Erk1/2,ras C,PKBA,Pia A and PI3K(1-5)play a role in EF guiding cell migration.Among them,Gγ subunit is important for promoting effects of caffiene in EF guiding cell migration in Dictyostelium discoideum.Caffeine may activte Erk1/2phosphorylation through Gγ subunit to promote EF guiding cell migration.The TORC2/PKB/PI3 K signaling pathway may be also involve in the promoting effect of caffeine in EF guiding cell in Dictyostelium discoideum. |
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