Experimental Structural Characterization and Md Simulation of F-Actin Self-Assembly
Nguyen, Lam Thanh (author)
Hirst, Linda S. (professor co-directing dissertation)
Xiong, Peng (professor co-directing dissertation)
Steinbock, Oliver (university representative)
Piekarewicz, Jorge (committee member)
Van Winkle, David (committee member)
Berg, Bernd (committee member)
Department of Physics (degree granting department)
Florida State University (degree granting institution)
A study has been carried out on F-actin self-assembly to address various questions regarding the mechanisms for assembly and the properties of the assembled system. We are interested in two Factin self-assembly mechanisms, counter-ion induced assembly and F-actin assembly in the presence of cross-linking proteins. In the first mechanism, previous studies have been carried out to understand how like-charged biopolymers can form bundles and what conditions satisfy bundle equilibrium revealing the interesting physics behind F-actin self-assembly. Our study aims to elucidate the structure of assembled F-actin by using a combination of different structural characterization techniques, including fluorescence microscopy, transmission electron microscopy (TEM) and small angle x-ray scattering (SAXS). Based on the x-ray data we find that as counter-ion concentration is varied, F-actin forms two bundle phases at low and high counter-ion regimes. In the high counter-ion regime, our x-ray data reveals a hexagonal bundle structure that is consistent with results seen in a previous study. In the low counter-ion regime, we propose a hexagonal bundle structure in which each elementary unit is composed of two actin filaments coupled together. This model is supported by our TEM evidence and can explain the gradual structural transition as the counter-ion concentration is varied. It has been shown in previous studies that in the presence of cross-linking proteins F-actin can form fascinating structures, such as a hierarchical network structure in the presence of alpha-actinin protein. To understand the mechanism for cross-linker-induced F-actin assembly we carry out a molecular dynamics (MD) simulation study, primarily based on the alpha-actinin/F-actin system. Our simulations are able to reproduce the hierarchical network structure observed experimentally. Calculation of the Fourier transform of the mass density of the simulated system yields results that match those of x-ray scattering on the real system very well. Based on these preliminary simulation results, we carry out a systematic study using a combination of confocal fluorescence microscopy and MD simulations to reveal the role of different controlling parameters in the F-actin assembly. We find that, F-actin can form various structures as we vary different parameters, including the absolute actin concentration (CA) and F-actin length (L), the cross-linker/actin molar ratio, gamma, and morphology of the cross-linker. In the presence of alpha-actinin in the high gamma regime, F-actin can form homogeneous and inhomogeneous bundle networks by varying CA and gamma. MD simulations show that, the assembled alpha-actinin/F-actin can change from a single filament network to an individual bundle phase as L is varied. In addition, as the cross-linker morphology is varied to represent filamin and fascin proteins, our MD simulations can reproduce characteristics of the real systems. Compared with F-actin bundles formed in the presence of alpha-actinin, bundles formed in the presence of filamin do not have an ordered internal structure. This is revealed by MD simulation observations and supported by an x-ray scattering study on the real system.
MD simulation, SAXS, self-assembly, cytoskeleton, F-actin
March 29, 2011.
A Thesis submitted to the Department of Physics in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Linda S. Hirst, Professor Co-Directing Dissertation; Peng Xiong, Professor Co-Directing Dissertation; Oliver Steinbock, University Representative; Jorge Piekarewicz, Committee Member; David Van Winkle, Committee Member; Bernd Berg, Committee Member.
Florida State University
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