Phase Separation And Shape Transformation Dynamics Of Muti-Component Vesicles

Biomembranes are mainly composed of lipid bilayers and membrane proteins. The lipid bilayers are multi-component fluid membrane in which phase separation could occur, resulting in "Raft" structure that are in biophysical process such as signal transduction and membrane trafficking pathway. Therefore, it is helpful to understand the structure and function of biomembranes through studying lipid phase separation in the bilayers. However, real biomembranes are complicated, it necessary to construct a simple model membrane to simplify the problem. Based on the "Raft" component found in biomembranes, we have used the vesicles composed of DOPC/BSM/Chol as the model membrane. The domains have been observed by fluorescent microscope during the phase separation, in which the CCD camera provides an effective means to track the dynamic process.This thesis is stimulated by the biomembranes phase separation. We mainly pay our attention to the non-equilibrium behaviors of the multi-component membranes such as domain dynamics, shape transformation dynamics induced by domains or detergents, which is accomplished by using microscope and micromanipulator. The main contents are summarized in the following:(1) The DOPC/BSM/Cholesterol system was used as a model membrane. After decreasing the system temperature below the miscibility temperature, rich phase separation and domain growth behaviors of the vesicle including both interconnected structure and droplet structure were observed. The influence of temperature, composition, hydrodynamics, dye probe and area-to-volume ratio on the phase behavior were discussed. The coarsening process of the domains was divided into two stages:in the early stage, flat domains coalescence each other and the number of domains follows an exponential law:N～t-2/3; but in the later stage, flat domains grow large enough to bulge into hemispherical caps by CIC (collision-coalescence mechanism or collision-induced collision mechanism) The number of domains decayed with an new exponential law:N～t-2. (2) The process of phase separation and coupled shape transformation dynamics mediated by the domains were investigated. Firstly, whether domains will become the buds determined by the bending rigidity, line tension, normal pressure difference, area-to-volume ratio and the thickness of membrane etc. The shape transformation is mainly driven by the line tension of the domain edge, but the spherical vesicle will not budding because of fixed area. Even in the late stage of phase separation in which the Lo and Ld are separated thoroughly into two parts, vesicles seemed to not reaching in a final equilibrium state yet. There are two possible ends of the phase-separated vesicles: (ⅰ) the membrane will rupture into the two smaller vesicles; (ⅱ) the long tubular buds will grow gradually from the Lo phase. All of these cases are accompanied with the change of osmotic pressure, which leads to the water leak out of the inner of the vesicle to the outer.(3) To study the influence of Lyso-PC on the vesicles in different stages of phase separation, we prepared the giant unilamellar vesicles (GUV) by electroformation. The GUV is big enough so that we can micromanipulate on it. Above the miscibility temperature, the Lyso-PC will lead to the shrinkage of the vesicles after it is added around the vesicles using microinjection. When the concentration of Lyso-PC is low, the vesicle will shrink smoothly. During this stage, we find that the radius of vesicles will decrease with time at first, following a scaling law R～t-0.25, then after the certain time, the radius of the vesicles will not change further because of the low concentration. On the hand, the vesicles will shrink sharply with high concentration of Lyso-PC. Similarly, the radius of the vesicles decay with the time following the same law R～t-0.25, but the law change into R～t-1 in the late stage. A theoretical model based on pore nucleation, open and close was used explain the shrinkage dynamics of the vesicles. Furthermore, the shape transformation of vesicle induced by Lyso-PC below the miscibility temperature was investigated. In this situation, the vesicles were not destroyed by Lyso-PC. On the contrary, the dark domains (Liquid-Ordered) which are "Raft-like" begin to bulge so as to budding with high concentration of Lyso-PC. We explained the phenomena by Area-Difference-Elasticity model.(4) In the presence of Ca2+, the dynamic hexagonal pattern emerged below the miscibility temperature of the vesicle composed of lipid/cholesterol. This phenomenon is the same for a variety of lipids with different lengths of saturated acyl chains or different hydrophilic heads. Cholesterol is believed to play an important role in forming this kind of pattern. Cholesterol as a flat, rigid molecule can interact with the carbonyl by hydrogen bond, which will increase the mobility of the acyl chains, then the lateral pressure increased. Consequently, cholesterol exerts a compression effect on the area per lipid molecule that becomes more subtle to the Ca2+. Under the low temperature, Ca2+ can approach four phosphates close enough to form linear network which may facilitate the lipid change from the layer phase to the cubic hexagonal phase. The evolution of pattern determined by the competition of long-range electrostatic and line energy. The line tension became more obvious in the late stage of phase separation in which the hexagonal domain change to the circular domain. If one further increases the concentration of Ca2+, the shape of the domain will keep the hexagonal-like. Then we can conclude that the electrostatic play an important role in the formation of the hexagonal pattern. In addition, instead of Ca2+, Na+ and Al3+ have been used and the similar phenomena were found. Finally, we are sure that such kind of phase separation is a reversible process, because the hexagonal pattern can be manipulated to happen or disappear by increasing or decreasing temperature.