Fabrication And Functional Study Of Phospholip Based Protoorganelles And Self-powered Artificial Cells

The construction of protoorgranelle and artificial cell models can provide simplified and controllable models for studying life activities of real cells.Protoorganelle and artificial cell models can provide stable environments and independently controllable confined spaces for complex biochemical reactions in cells.The research goals of protoorganelle and artificial cell are to mimic cells in terms of structures and functions,so as to build minimal cells from the bottom up.Currently,the research on protoorganelles is mainly based on spherical organelles,and the morphological mimicry of organelles such as Golgi apparatus,endoplasmic reticulum and thylakoid has not been realized.In addition,the construction of self-powered artificial cells is also one of the key issues in the field.At present,the construction of self-powered artificial cells is mainly based on the application of ATP synthase with complicated operation procedure.In the field of artificial cells,it remains a challeng to construct cytoskeleton in artificial cells trigered by internal energy.We constructed the phospholipid assembly with cisternae stacking structure and studied the material transfer between artificial organelles.By preparing sustainable self-powered artificial cells,the construction of cytoskeleton was realized,and the reversible deformation of muscle-like tissue were simulated.Phospholipid bicelles with square holes were prepared by programmed cooling in ethanol/water solution.Using scanning electron microscope,it was clearly observed that the overall shape of the phospholipid bicelles was circular with a square hole in the center.The influence of the concentration of phospholipid molecules,the cooling rate,the ratio of ethanol to water and the type of alcohol on the diameter,hole edge/diameter ratio and thickness of the phospholipid bicelles were further studied.The phospholipid bicelles were dispersed in the aqueous solution to form a cisternae stacked structure similar to the structures of mitochondrial inner cristae,endoplasmic reticulum and Golgi apparatus.An protoorganelle with catalytic function was constructed by encapsulating the macromolecular enzyme proteins in a cisternae stacking structure using the interaction between biotin and avidin.Through electrostatic interaction,small phospholipid vesicles and cisternae stacking structures fused to realize the transfer of enzymes between protoorganelles.Mitochondria were extracted from cells,and the purity and morphology of the extracted mitochondria were characterized.The activity of isolated mitochondria in buffer solution maintained for at least 36 hours.Under optimal buffer conditions,21μmol/L,12μmol/L and 3μmol/L pyruvate yielded 0.033×10^-3 mol/L,0.026×10^-3mol/L and 0.009×10^-3 mol/L ATP by the equal amounts of mitochondria,respectively.The mitochondria were encapsulated in phospholipid vesicles by electroformation and emulsion methods.The advantages of the two methods for preparing artificial cells were compared.The pyruvate molecules entered the artificial cells through the melittin pores,and stimulated the mitochondria in the artificial cell to produce ATP.Artificial cells with self-powered function were prepared.Artificial cells containing mitochondria can generate ATP through respiration by adding pyruvate.ATP molecules initiate the polymerization of actin monomers to form actin filaments.The controlled polymerization of low-concentration actin monomers in artificial cells was achieved using ATP molecules.The polymerization of actins monomers into actin bundles was studied via the use of methylcellulose.Under the crowding effect of methylcellulose,actins polymerized on the inner surface of the artificial cells,and the artificial cells were induced to change from pherical to spindle shape.Under laser irradiation,the actin bundles were depolymerized,and the artificial cells recovered from spindle to spherical shape.Through the acoustic field and the semi-fusion between artificial cells,linear cell colonies with artificial cells were obtained.Under pyruvate and laser stimulation,linear cell colonies exhibited collective contraction and relaxation behavior.Every artificial cell in the colony expanded and contracted synchronously.The time were about 1300 s,1920 s and 2400s respectively,when the artificial cell colonies composed of 2 GUVs,3 GUVs and 4GUVs reached the maximum deformation.When the GUVs colonies were in the state with maximum deformation,the angles between the long axes of each GUV were close to 0°,suggesting that each GUV deformed in the same direction.