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Actin-based cell motility plays crucial role throughout the lifetime of an organism. While the dendritic nucleation model explains the initiation and organization of the actin network in lamellipodia, two questions need to be answered. In this study, I reconstructed cellular motility in vitro to investigate how actin filaments are organized to coordinate elongation and attachment to leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel beads or thin glass nanofibers coated with N-WASP WCA domains. During sustained motility, physiological concentrations of Mg²⁺ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg²⁺ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, filament capping, or actin shell formation. Addition of other types of crosslinkers restored both comet tail attachment and particle motility. We propose a model in which polycation-induced filament bundling sustains processive barbed end attachment to the leading edge. I lowered actin, profilin, Arp2/3, and CP concentrations to address the generation of actin filament orientation during the initiation of motility. In the absence of CP, Arp2/3 nucleates barbed ends that grow away from the nanofiber surface and branches remain stably attached to nanofiber. CP addition causes shedding of short branches and barbed end capture by the nanofiber. Barbed end retention by nanofibers is coupled with capping, indicating that WWCA and CP bind simultaneously to barbed ends. In pull-down assays, saturating CP addition only blocks WWCA binding to barbed end by half. Labeled WWCA bound to barbed ends with an affinity of 14 pM and unlabeled WWCA with an affinity of 75 pM. CP addition increased WWCA binding slightly at low CP concentrations and decreased WWCA binding to 50% at high CP concentrations. Molecular models of CP and WH2 domains bound respectively to the terminal and penultimate actin subunit showed no overlap and that CP orientation might blocks WWCA dissociation from the penultimate subunit. Simultaneous binding of CP and WWCA to barbed ends is essential to the establishment of filament orientation at the leading edge.

Gas phase kinetics of the reactions involving hydroxyl radical and hydrogen atom were studied using experimental and ab initio theoretical techniques. The rate constant for the H + H2S reaction has been measured from 298 to 598 K by the laser photolysis/resonance fluorescence (LP-RF) technique. The transition state theory (TST) analysis coupled with the measurements support the suggestion that the reaction shows significant curvature in the Arrhenius plot. The LP-RF technique was also used to measure the rate constant of the H + CH3Br reaction over the temperature range 400-813 K. TST and density functional theory (DFT) calculations show that the dominant reaction channel is Br-abstraction. The reaction H + CF2=CF-CF=CF2 was first studied by flash photolysis/resonance fluorescence (FP-RF) method. The experiments of this work revealed distinctly non-Arrhenius behavior, which was interpreted in terms of a change in mechanism. DFT calculations suggest that the adduct is CF2H-CF•-CF=CF2. At lower temperatures a mixture of this molecule and CF2•-CFH-CF=CF2 is likely. The theoretical calculations show that H atom migrates in the fluoroethyl radicals through a bridging intermediate, and the barrier height for this process is lower in the less fluorinated ethyl radical. High level computations were also employed in studies of the rate constants of OH + chloroethylenes reactions. VTST calculations indicate that, except the reaction of OH + C2Cl4, these reactions present a complex behavior. For OH + C2Cl4, conventional TST calculation shows a simple positive temperature-dependence behavior.