Osmotic Gradient Coordinated Spontaneous Curvature Mediated Phospholipid Redistribution In Cell-Like Membrane

Giant unilamellar vesicle membrane is an ideal model of cell membrane,analogous to the self-enclosed lipid matrix of the plasma membrane of all living cells.Currently,giant unilamellar vesicles are used to explore several aspects of bio-membranes that can be directly observed under an optical microscope.Such as,the membrane permeation triggered by the added compound,the lateral lipid heterogeneity,or the membrane budding and fission.The paper is based on a cell-like membrane model adopting the technique of electroformation to prepare the giant unilamellar vesicles.We apply a highly sensitive inverted fluorescence microscope characterization technique combined with a fluorescence leak experimental characterization technique.The paper presents a study of osmotic gradient coordinated spontaneous curvature mediated phospholipid redistribution in cell-like membrane.Osmotic pressure can break the fluid balance between intracellular and extracellular solutions.In hypo-osmotic solution,water molecules,which transfer into the cell and burst,are driven by the concentration difference of solute across the semi-permeable membrane.The complicated dynamic processes of intermittent bursts have been previously observed.However,the underlying physical mechanism has yet to be thoroughly explored and analyzed.In the chapter3,section 1,the intermittent release of inclusion in giant unilamellar vesicles was investigated quantitatively,applying the combination of experimental and theoretical methods in the hypoosmotic medium.Experimentally,we adopted a highly sensitive electron multiplying chargecoupled device to acquire intermittent dynamic images.Notably,the component of the vesicle phospholipids affected the stretch velocity,and the prepared solution of vesicles adjusted the release time.Theoretically,we chose equations and numerical simulations to quantify the dynamic process in phases and explored the influences of physical parameters such as bilayer permeability and solution viscosity on the process.It was concluded that the time taken to achieve the balance of giant unilamellar vesicles was highly dependent on the molecular structure of the lipid.The pore lifetime was strongly related to the internal solution environment of giant unilamellar vesicles.The vesicles prepared in viscous solution were able to visualize long-lived pores.Furthermore,the line tension was measured quantitatively by the release velocity of inclusion,which was of the same order of magnitude as the theoretical simulation.In all,the experimental values well matched the theoretical values.Our investigation clarified the physical regulatory mechanism of intermittent pore formation and inclusion release,which provides an important reference for the development of novel technologies such as gene therapy based on transmembrane transport as well as controlled drug delivery based on liposomes.Giant unilamellar vesicle is the simplest cell-like closed compartment.Although water molecules are easily balanced on the vesicle barrier,but the passive diffusion of solute is strongly blocked.Hence it is easy to establish an osmotic gradient between the closed vesicles and the surrounding free bath,which triggers osmosis.In the Chapter 3,Section 1,we studied the regulation of intermittent release of single-component GUVs in hypo-osmotic media.However,given the diversity of human cells,and the multi-component membranes coupled with the milder osmotic gradients that can occur in living cells.In the Chapter 3,Section 2,based on the giant unilamellar vesicle,we analyzed the phospholipid redistribution behavior of multi-component cell-like membrane mediated by osmotic gradient with experiment combined with theory.In the experimental study,we selected a ternary lipid mixture of cholesterol(Chol),sphingomyelin(SM)and unsaturated phospholipids(POPC)to form GUVs.Osmotic gradient established by deionized water.Multi-component membrane surface appears to flip between a state characterized by large microscopic domains(phase separation)and an optically homogeneous state(homogeneous phase).Moreover,we observed transient pores of the time cascade,according to the release process of the daughter vesicles through the pore,we analyzed the underlying biophysical mechanism in the theoretical study.The pore lifetime was calculated to be sufficient to allow partial solute leakage.The cycle pattern indicated quasi-steady state self-regulatory behavior,sensing and regulating its microenvironment in a negative feedback loop.A remarkable property of sugars(such as glucose and sucrose)is that they protect biomembranes from dehydration damage,which indicates potential sugar-lipid interactions.In low-hydration conditions,the lipid bilayer-sugar interactions have been extensively studied.In the Chapter 4,we studied fully hydrated lipid membranes exposed to two sugars(glucose and sucrose)based on a giant unilamellar vesicle.In experimental studies,when the membrane inner solution is filled with sucrose(/glucose)and the outer solution is filled with glucose(/sucrose),the sugar asymmetry causes spontaneous bending of the phospholipid bilayer.It can generate outwardly budding(/inwardly budding)vesicle shapes in which the direction of bending can be reversed by exchanging internal and external solutions.The results showed that the sugar-lipid interactions induced a significant positive spontaneous curvature(/negative spontaneous curvature),which was caused by effective repulsion and attraction of the sugarlipid interactions.In theoretical studies,we will try to obtain a range of parameters which stabilize the membrane shape by two dimensionless physical parameters,including the volume to area ratio and the normalized spontaneous curvature,using Matlab software simulations,and discuss possible molecular mechanisms.The detailed comparison between theory and experiment shows that the inward budding(/outward budding)shape of vesicles remains stable within a wide range of parameters.There is significant overlap between the different stability regimes,reflecting the dramatic free energy landscape in shape space.Furthermore,the phase diagram was obtained to help determine the pathways of cell-like membrane shape transitions(experimentally we collected two possible pathways to outward budding vesicles as well as one possible pathway to inward budding vesicles),providing us with an experimental and theoretical basis for in-depth studies to open and close membrane necks of these shapes in a controlled and dependable way.Finally,we summarize the conclusions of the research contents of this paper obtained and provide an outlook on future research interests.