Many important cellular functions depend on dynamic actin networks forming at the correct location and time during the cell cycle. Cellular division and motility are examples of processes in which actin filaments must be nucleated, polymerized, and disassembled with spatiotemporal precision. Here, I investigate molecular mechanisms of various actin binding proteins (ABPs) and how they are affected by other ABPs and actin binding toxins. The main project focuses on Ena/VASP, which are tetrameric actin elongation factors that continuously bind F-actin barbed ends while increasing their elongation rate within dynamic bundled networks such as filopodia. We tested how Ena/VASP's molecular mechanism is affected by the presence of different bundling proteins. We used single-molecule TIRFM and developed a kinetic model to dissect Ena/VASP’s processive mechanism on bundled filaments. The results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple filaments is critical for this process. I investigated molecular mechanisms of various ABPs during multiple collaborations, many which focused on bundling proteins that I have studied in relation to Ena/VASP. I found that fascin, the main bundling protein in filopodia and enhancer of Ena/VASP processivity, also plays a role in reducing Arp2/3 complex-mediated branching. We also showed that fascin sorts with alpha-actinin due to an intrinsic mechanism dictated by filament spacing. We visualized this sorting by electron microscopy and observed the transition between a fascin and alpha-actinin domain. Further investigation into alpha-actinin's bundling mechanism showed that tropomyosin increases alpha-actinin dynamics. My other collaborations focused on the pathogenic Vibrio bacteria. We found that ACD actin oligomers inhibit Ena/VASP elongation and cause Ena/VASP to cap filaments. Furthermore, we found that Vibrio nucleation factors VopL/F nucleate filaments from pointed ends of F-actin. Overall, my work has shown the importance of not only characterizing molecular mechanisms of ABPs, but also how these ABPs work in concert with the multiple other ABPs that are found within the cell. The gained knowledge from these studies are a step forward for the field to fully grasp the role each ABP plays in the larger system of the actin cytoskeleton.



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