Mechanisms Of Receptor-mediated Endocytosis Affected By Cell Membrane Tension

Targeted drugs in cancer therapy,such as nanoparticles,are internalized by cells through receptor-mediated endocytosis.However,nanoparticles are inefficient in endocytosis and not specific enough in invasion of targeting cells.In order to improve the success rate of nanoparticle uptake,it is of great scientific significance and engineering value to study the mechanical mechanism of endocytosis.By combining numerical method and experimental observation,the effects of mechanical properties of nanoparticles and cells on endocytosis success rate were analyzed,as well as the relationship between the size of endocytotic vesicles and the cell membrane tension was investigated in this thesis.Firstly,in the passive endocytosis model,assuming the energy was provided only by the chemicals energy that released by receptor-ligand bonds formation.The system composed of nanoparticle,receptor-ligand bonds and cell membrane was established,and the minimun value of the system deformation energy function was searched by optimization method.The shape of the system in quasi-static state was obtained.The effects of nanoparticle size and stiffness,the binding strength of receptor-ligand bonds,and the stiffness of cell membrane on the maximum wrapping fraction of endocytosis were analyzed.Results showed that the binding strength of receptor-ligand bonds and the nanoparticle size have significant effects on passive endocytosis,while the effects of nanoparticle stiffness and cell stiffness are not so significant.This indicates that the binding energy and adhension force between targeted molecules modified on drug surface and receptors specifically expressing on cell membrane surface should be considered first in drug design,followed by the size of the drugs.Two criteria for passive endocytosis are proposed:1)The bonds need to be sufficiently strong;and 2)Enough bonds per unit area to provide sufficient energy.Passive endocytosis cannot be completed when either the energy or force provided by the receptor-ligand bonds is insufficient.Therefore,the completion of endocytosis requires the cells to provide extra energy and force through active mechanisms.Secondly,due to the natural existence of clathrin-coated pit(CCP)structure inside the cells to assiste the receptor-mediated endocytosis,we extended the passive endocytosis model to the active endocytosis model considering CCP and analyzed the effect of cell membrane tension on the mature size of CCP.Results show that the force provided by receptor-ligand bonds can hardly guarantee full wrapping of the nanoparticles especially under tension conditions,thus the receptor-ligand bonds were not the most important factor to determine whether endocytosis be completed or not.An interesting finding is that the size of CCP at the final wrapping step increases with the increase of cell membrane tension,and the diameter of necking part of cell membrane also increases.When the bonds between nanoparticle and cell membrane are all broken,and the tension of the cell membranes is high enough,the nanoparticles may escape from the "bottle neck" of CCP.When considering the tension of cell membrane,the diameter of endocytic vesicles depends on not only the size of nanoparticles,but also on the balance between cell membrane tension and CCP reinforcement.Thirdly,in order to verify the CCP size increasing with the increase of cell membrane tension in the numerical finding,we observed the size and growth time of CCPs in natural state.The fixed cells were scanned by structured illumination microscopy in 3D,thus the size and spatial position of CCPs were observed.The CCPs lifetime of living cell were observed by total internal reflection fluorescence microscope microscope.Through K-S test,the generation intensity of CCPs was best fitted by the normal distribution.The process of CCPs vesicle formation,and then maturation or abortion is reproduced numerically by Monte Carlo simulation.By comparing with the experimental statistical results,the reliability of Monte Carlo scheme was verified,and the CCPs lifetimes satisfies the exponential distribution was tested.In addition,it revealed that the growth time of CCPs is proportional to the equivalent volume of CCPs.The results showed that the CCPs of the size larger then 250nm are belong to the mature group,and this finding is used to further analyze the relationship between the mature size of CCPs and the membrane tension.Fourthly,the standard cell-spreading model was obtained by 3D reconstruction of cell morphology according to the spatial distribution of CCPs.Combining the particle method with the energy minimization method,the numerical solution of cell spreading shape in steady state was found.The tension distribution on the cell membrane surface was obtained,which present a gradual trend of higher tension at the bottom circumference and lower tension on the top of the cell.The numerical results were qualitatively compared with the previous experimental results,which verified the reliability of the numerical simulation.Through statistical analysis,it was found that the number and density distribution of CCPs along cell height were positively correlated with the cell membrane area expansion rate.This result supports in the numerical finding that the size of CCPs increased with the increase of cell membrane tension at the final wrapping step.CCP can help to maintain the stability of endocytic vesicle structure,thus the larger CCP structure is needed to overcome the higher energy barrier caused by cell membrane deformation when the cell membrane tension.