Kinetic and kinematic properties of actin network assembly and nonlinearity of cross-linked fibrin gels.

摘要

Although recent studies have identified the biochemical components required to generate motile forces by actin polymerization, how the forces are generated remains unclear. To elucidate this molecular mechanism of cell motility, I investigated the biophysical conditions under which actin-based growth and motility take place.;In Part I of this thesis, I report an experimental study on the kinetics of actin assembly mediated by branching and capping proteins. The experiments were performed by fluorescence imaging and light-scattering intensity measurement. The findings confirm the recent theoretical prediction that a "branching explosion" occurs during polymerization. Furthermore, the branching explosion occurs over a limited range of the ratio between branching protein and capping protein concentrations. This is also consistent with the theoretical model. These results establish a natural link between the kinetic theory of actin assembly in vitro and the cytoskeletal structure and actin dynamics in motile cells.;In Part II of this thesis, I present an in vitro study of the actin-based movement of functionalized spherical beads in comparison with those of bacteria like Listeria. Long trajectories induced by the spherical beads show characteristic differences with those observed for bacteria, which have both an elongated shape and an asymmetric expression of the polymerization inducing enzyme. The experimental trajectories can be simulated using a generalized kinematic model that includes the rotation of the bead relative to the actin tail. These results imply that the trajectories of spherical beads are mechanically deterministic rather than random, as suggested in stochastic models.;In Part III of this thesis, I examine nonlinear viscoelasticity of cross-linked biopolymer networks, by rheological measurements of shear moduli and normal stresses in fibrin gels. The results for coarse fibrin gels are consistent with expectations from theories of rod-like filament networks. Comparison of rheological and optical properties shows that the filament alignment, as measured by optical retardance increases with increasing shear strain but lags behind the increase in shear moduli.