Investigation On The Actin Polymerization At The Water/Solid Interfaces And Its Mechanical Force Effects

Globular actin(G-actin) can assemble into fibrous actin(F-actin) under certain physiological conditions, while F-actin can dissemble into G-actin unde certain conditions. The conversion of the two actins plays a key role in the cytoplasmic division, cell migration, endocytosis and intracellular substances transportation. On one hand, it is well known that the mechanical force can induce the complex signal transduction and biochemical reaction in the cell, which can cause the change of the actin network. The intracellular actin molecules are able to response to the mechanical stimulate through the polymerization and depolymerization. However, the mechanisms have still not been well understood so far. On the other hand, actin is important for the transport of materials in a cell by ascting as an orbit of a motor protein, myosin. Recent studies have indicated that, as the orbit of protein motor, F-actin can be used for design and construction of functional nano-bio-machines, which will have great potential in the material transportation at the micrometer and nanometer scales. Since the artificial nano-bio-machines will be operated on different substrates, it is very important to investigate actin assembly behaviors on different substrates.In order to address the abovemensioned problems, this thesis has carried the following three works by employing atomic force microscope(AFM).Firstly, the assembly processes of actin on mica substrate and lipid membrane by real time imaging of atomic force microscope and laser scanning confocal microscope were studied. It was found that on the bare mica substrate, the small amount of G-actin nucleating points formed and subsequently self-assembled into long actin filaments with lengths up to tens of micro-meters. While on the positively-charged lipid membrane(DPPC: DPTAP = 4:1), large amounts of nucleating points quickly formed, leading to the formation of relatively shorter filaments. The proposed optimum condition for exploring actin assembly process with AFM in vitro is positively-charged substrates should be used while the AFM imaging force should be lower than 50 pN. These results laid foundation for construction of micro- and nano-scale transport systems based on actin.Secondly, the effect of physical forces on the actin polymerization and depolymerization behaviour were investigated in situ by utilizing the tapping mode AFM in liquid. Our results showed for the first time that actin could react to physical forces directly without actin related proteins(ARPs) in vitro. G-actin was boosted to polymerize with minimal force but depolymerize with strong physical force. This result should be helpful for understanding the response of actin to mechanical stimulates.Thirdly, we have explored the possibility to construct the orbit of protein motor by physically manipulating F-actin on solid substrates by using the Molecular Combing, the PDMS stamp, and AFM nanomanipulation techniques. Experiments showed that the molecular combing technique in general can be used for align actin filaments on the solid substrates. In addition, AFM nanomanipulation showed the ability to cut precisely the F-actin at the nanoscale.