Separation Of Caveolae And Atomic Force Microscopy Study And Simulation Study In Vitro On Them

Caveolae were spherical,elliptical or flask-shaped invaginations(50-100nm in diameter) on the surface of cell plasma membrane.They can also form separated vesicles or tubal structures,or can extend on plasma membrane,and some times can be fused into special structures more than 100nm in diameter.Caveolae widely existed in different cells,especially in endothelial cells,adipocyte, vascular smooth muscle cells,fibroblasts,and lung epithelial cells,and so on.They played important roles in many biological processes,such as endocytosis,pinocytosis,transcytosis,internalization of bacteria and viruses,cholesterol transport and regulation,maintenance of intracellular Ca²⁺ steady balance,protein sorting,and signal transduction,etc.In this paper,we researched caveolae in two aspects:(1) separation of caveolae and study on them using atomic force microscopy(AFM);(2) preliminary study on simulation of caveolae in vitro. These studies laid a good foundation for the further study on the structure and functions of caveolae, and had important significance.The experimental results are as follows:(1) Separation of caveolae and study on them using AFM:We separated caveolae from rat lungs by floating plasma membrane treated with ultrasonic in OptiPrep gradient(a detergent-free method). After twice ultrasonications at 0℃,caveolae were detached from other part of plasma membrane, and then by several density gradient centrifugations,caveolae were separated because of their unique buoyant density.This method of separation was simple,feasible and mild.Using this method we conquered the disadvantage that some special proteins would be removed by traditional detergent method and we separated intact caveolae close to real situation in cells.Then we observed them by atomic force microscopy(AFM) in different conditions and the best AFM images of caveolae were obtained by diluting them 500 times with deionized water.(2) Preliminary study on simulation of caveolae in vitro:in order to study the mechanism of the interaction between different kinds of lipid which constituted caveolae,and in order to lay a foundation of the further research of simulation in vitro,the phase behavior and morphology of mixed binary systems of phosphatidylcholine(PC) with stearic acid(SA) were investigated at the air /water interface by surface pressure-area(Ï€-A) isotherm measurements and by AFM observation. The analyse of theÏ€-A isotherms showed that the condensed states of the binary systems changed with SA molar fractions(X_SA) at different surface pressures at 30℃.TheÏ€-A isotherms were distributed between theÏ€-A isotherms of pure PC and SA regularly,which was proved by Gibbs energy analyses theoretically,and was directly confirmed by AFM observation.This part studied on the phase behavior and morphology of mixed binary systems of PC/SA at the air/water interface by experiment researches and theoretical researches,so this study had important meaning of exploration.This paper provided important experimental basis and new thought for the further study on the structure and functions of caveolae,and had much significance.