Title: Assembly in Dynamic Nanoscale Systems
Description: Biological systems are intricate self-assembled systems built from dynamic nanoscale components. These nanoscale components are responsible for many tasks, from subcellular (e.g. DNA replication, cytoplasmic streaming, intracellular transport) to organismal (e.g. intercellular signalling, blood circulation). At each level, biological materials demonstrate complex and dynamic behaviors which are still robust to many perturbations, requiring a balance of dynamism and stability. Being able to emulate biology by dynamically assembling complex systems and structures from nanoscale building blocks would greatly expand the types of materials and structures available, possibly leading to better smart, adaptive, self-healing materials in engineering. The overarching goal of this dissertation is to further the understanding of assembly in dynamic nanoscale systems. To this end, in vitro assays of kinesin motor proteins and microtubule cytoskeletal filaments are employed, providing a well-tested, minimalist, and convenient model system.

In these assays, the kinesin motors are attached to the surface of the flow cell and the microtubule filaments are propelled over them. As the majority of past studies in active self-assembly of microtubules have been performed with biotin-labeled microtubules with streptavidin as a cross-linker (a "sticky" gliding assay), the first three parts of this dissertation focus on that system. In the first part, the adsorption kinetics of the streptavidin cross-linker onto the microtubule, which determines the interaction strength between microubule building blocks, is studied. The adsorption curve suggests that this is a negatively cooperative process, and here, the cause of the apparent negative cooperativity in the adsorption process is elucidated as a combination of steric and electrostatic interactions. In the second part, the difference between kinesin-propelled assembly and diffusion-driven assembly is investigated. While the kinesin-propelled microtubule assay has been used for over a decade, a control experiment comparing the active motor-driven system to a passive diffusion-driven system had never been performed.

The control experiments showed conclusively that the passive system resulted in smaller and more disordered structures. Furthermore, these results fit well with existing models. The third part investigates the origins of microtubule spools observed in kinesin-propelled microtubule gliding assays, where the microtubules are allowed to cross-link via streptavidin and biotin. These microtubule spools have long been considered an example of a non-equilibrium structure which arises in motor-driven assembly. These spools exist in a dynamic state, having been observed to unwind in previous studies, and store large amounts of bending energy. Determining the origins of these spools is a first step towards understanding how to induce dynamically stable states. Finally, in the last part, a new dynamic system is engineered in which the microtubule assembles its own kinesin track as it moves along the surface while kinesin tracks which are not being used spontaneously disassemble. Thus, this system is stable enough to promote the motion of microtubules over the surface, but dynamic enough to allow for components to be recycled and assembled as needed.

While such systems have been realized with mesoscopic to macroscopic components, such a system had not been realized in the nanoscale. As such, the realization of this system is the first step towards designing biomimetic active materials. Throughout this dissertation, the importance of short-range interactions on assembly kinetics is highlighted. The findings presented not only further the understanding and theory behind self-assembly in active nanoscale systems, but also further push the boundaries of experimentally realized systems.