Cryo-EM Studies of Microtubules and Bacterial Nanocompartments

This dissertation focuses largely on structure-function relationship of microtubules, with a supplemental focus on bacterial nanocompartments known as encapsulins./nMicrotubules (MTs) are an essential component of the eukaryotic cell. MTs are crucial for intracellular trafficking, cellular division, and motility, along with many other functions within the cell. The building blocks of a MT are tubulin heterodimers, which are GTPases that self-assemble to form a hollow, cylindrical MT architecture. The diversity of MT functions is achieved in part by a curious phenomenon known as dynamic instability whereby MTs are in a constant state of flux between growth and catastrophic depolymerization. These dynamics are directly linked to the nucleotide state of the MT, whereby the interplay the GTP and GDP nucleotide states determines the propensity for growth or shrinkage. The intrinsic regulation of dynamic instability, in addition to extrinsic regulation by various MT-associated proteins (MAPs), is absolutely critical for proper cellular function./nCryo-electron microscopy (cryo-EM) was used to directly visualize these important biological assemblies in their native states. The first aspect of my work was to determine how the nucleotide state or the binding of common MAPs (EBs and Kinesins), affected the MT structure. Previous MT structures required MAP-binding to act as fiducials in order to facilitate high-resolution, and Chapter One of this dissertation shows the first high-resolution structures of undecorated MTs bound to various different nucleotides. The next aspect of my work was to examine the GTP-bound MT structure, in order to learn how certain MAPs specifically recognize the GTP state, and the conformational dynamics that occur upon GTP hydrolysis. Prior to this study, the MT field has used a GTP analog, GMPCPP, as a proxy for the GTP bound state. However, no one had actually observed MTs in the GTP state. Chapter Two in this dissertation uses mutated recombinant human tubulin that is hydrolysis-deficient to trap MTs that are truly GTP-bound. I found that these tubulin mutants are an invaluable platform for studying MTs. However, it appears that some mutants appear to create non-physiological assemblies, creating caveats for this system similar to the imperfections of nucleotide analogs./nThe last chapter of this dissertation is the culmination of work using cryo-EM to better understand the assembly principles for a bacterial nanocompartment known as the encapsulin. Encapsulins were first discovered in 2008, and are typically composed of an enzymatic cargo confined within a proteinaceous shell. This compartmentalization can provide the cell with protection from toxic intermediates, or help with increasing local concentration to make the reaction more efficient. The methods driving cargo encapsulation to support these diverse functions remained unknown. For one encapsulin species, I determined how the cargo enzyme was associated with the encapsulin shell, and found that encapsulin cargos do indeed use a form of symmetry-matching. In this experiment, I found that a pentameric cargo protein binds at the pentameric vertices of the encapsulin shell, thus regulating where the cargo is within the compartment as well as the stoichiometry of cargo encapsulation. Serendipitously, while purifying this encapsulin, I also realized it was a previously overlooked flavoprotein. Given the fact that the cargo enzyme is a ferritin-like protein performing redox chemistry, this flavoprotein designation is very intriguing and warrants additional experiments./nTaken together this dissertation utilizes cryo-EM as a tool to probe the structural underpinnings that drive microtubule and encapsulin assembly and function.

Advisor: Nogales, Eva