STRUCTURAL STUDIES OF THE MOLECULAR BASIS OF BRANCHING MICROTUBULE NUCLEATION

Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. 

Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. 

Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. 

To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish in vitro biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). 

Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from Pseudomonas aeruginosa can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.