Segregative Clustering of Lo and Ld Membrane Microdomains Induced by Local pH Gradients in GM1-Containing Giant Vesicles: A Lipid Model for Cellular Polarization

摘要

Several cell polarization processes are couplednto local pH gradients at the membrane surface. We haveninvestigated the involvement of a lipid-mediated effect in suchncoupling. The influence of lateral pH gradients along thenmembrane surface on lipid microdomain dynamics in giantnunilamellar vesicles containing phosphatidylcholine, sphingomyelin,ncholesterol, and the ganglioside GM1 was studied. LoLd phase separation was generated by photosensitization. Anlateral pH gradient was established along the externalnmembrane surface by acid local microinjection. The gradientnpromotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ldndomains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo andnLd phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processesnoccurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup.nLAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occursnwith decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ldnphase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering andnvesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. SuchnpH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients arenknown to be important, such as migrating cells and epithelial cells.