Study Of Microvilli,Cell Membrane Microfilaments And Their Applications By AFM

Cell membrane is the most important structural component of cells,which plays an important role in maintaining homeostasis and various physiological functions.However,some physiological functions cannot be performed without some special structures on the cell surface,such as microvilli,which play a vital role in nutrient absorption,immune regulation,signal transmission and other activities.Due to the limitations of traditional technologies,the relationship between the structural changes of microvilli on the nanoscale and the absorption function has not been directly confirmed under physiological conditions.Microfilaments are not only the supporting component of microvilli structure,but also an important component of cell structure.They are involved in the maintenance of cell morphology,cell adhesion,cell movement and other physiological functions.However,there are still many problems in the spatial distribution of microvilli and microfilaments in cells under the near physiological conditions.In addition,microfilaments participate in a variety of physiological activities induced by mechanical forces.However,whether microfilaments are involved in the transmission of autophagy signals between adjacent cells remains elusive.Atomic force microscopy(AFM)has a special advantage in the study of cell biology which can carry out high-resolution imaging of a variety of biological samples under physiological conditions and obtain the physical and chemical properties of the samples at the same time.It has been applied in the study of single molecule,cell and tissue samples.In view of the above problems,the dynamic changes of the microvilli and microvilli cluster were observed at the level of living cells by using high resolution imaging of AFM and precise control of force,combined with fluorescence imaging characteristics of fluorescence microscope.The relationship between the changes of microvilli structures and absorption function was studied on the nanometer scale under the near physiological condition.In addition,the role of microfilaments in mechanical force-induced autophagy signal transmission between adjacent cells was preliminarily investigated.The main contents are as follows:First,AFM high resolution imaging was used to observe the microvilli structure on the cell surface at the nanometer scale,and the size and morphological characteristics of single microvillus were obtained.In situ continuous imaging of living cells were carried out to observe the dynamic changes of single microvillus of two subtypes and microvilli clusters on the cell surface.It was found that the growth cycle of single microvillus and the phenomenon of the depolymerization and polymerization of microvilli clusters were observed.In addition,we also studied the direct correlation between the changes of microvilli structure and the absorption function of dextran under starvation and found that the structures of microvilli were dynamic reversible changes.Second,based on the characteristics of the precise control force of AFM,the distribution of the microvilli outside the cell membrane and the microfilaments inside the cell membrane were investigated.Without destroying the cell,the two-layer distribution of microvilli and microfilaments in the cell was studied.The surface layer was made of the bundle actin-filament forming the single and assembled clusters of microvilli emanating from the cell membrane;lying underneath this layer was an intricate network of thick and thin actin-filament beneath the cell membrane.In addition,the morphology and structure characteristics of single microfilament and the dense intracellular microfilament meshwork structures were obtained by AFM high resolution imaging.Thirdly,taking advantage of the precise control force of AFM,the role of microfilaments in autophagy signal transmission was also preliminarily explored.The probe of AFM was used to stimulate cells,and the effect of nano-mechanical force on autophagy was confirmed.It was found that autophagy induced by mechanical force could be transferred to adjacent cells,and such transfer could occur between cells of the same or different species.At last,by constructing the gap junctions of N2 a cells,the function of gap junctions in autophagy transmission between adjacent cells was studied.The above research contents demonstrated the advantages of AFM in studying the dynamic of living cells and the spatial distribution of actin-filament,as well as the functions of actin-filament in force signal transmission.At the end of the paper,the above work is summarized and prospected.