REGULATION OF MICROTUBULE DYNAMICS BY TUBULIN DIVERSITY AND SEVERING ENZYMES

Microtubules are cytoskeletal polymers that cycle through polymerization and depolymerization, a phenomenon known as dynamic instability. This behavior is essential for basic cellular functions such as cell division, migration, and morphogenesis. Microtubule dynamics is tightly regulated by two broad mechanisms: 1) intrinsically by tubulin isotypes or tubulin post- translational modifications and 2) extrinsically by various microtubule effectors. While many studies have helped us understand how effectors tune microtubule dynamics, we still do not have an understanding of how tubulin isotypes affect microtubule dynamics. This is because the majority of the microtubule dynamics studies have been performed with brain tubulin. Brain tubulin is heterogeneous, consisting of multiple tubulin isotypes and chemically diverse post- translational modifications making it impossible to understand the contribution of a single tubulin isotype or post-translational modification to microtubule dynamic properties. In the first section of my thesis, I explore the dynamic properties of a recombinant single human unmodified tubulin isotype, α1A/βIII. I also purified unmodified α1B/βI+βIVb tubulin from a stable human embryonic kidney cell line and found that this tubulin composition has dramatically different dynamic parameters than hetergenous brain tubulin. Moreover, I found that the addition of α1A/βIII tuned α1B/βI+βIVb tubulin dynamics proportionally. Finally, I explored how extrinsic factors such as microtubule severing enzymes can modulate microtubule dynamics. Severing enzymes break microtubules in an ATP-hydrolysis dependent manner. They are essential for the generation of microtubule arrays in neurons, the plant cortex, and spindle. Despite their discovery 30 years ago, their effects on microtubule dynamics has remained unknown. We found that severing enzymes extract tubulin dimers out of the microtubule lattice. These “holes” can be repaired by spontaneous GTP-tubulin incorporation along the microtubule shaft. This results in an increase in microtubule rescue frequency. Moreover, the newly severed ends emerge with a high-density of GTP-tubulin, which protects them from depolymerization. The combination of increased microtubule rescues and stable ends causes microtubule mass and number amplification. Together, my graduate work has shed light on how tubulin diversity tunes intrinsic microtubule dynamics and has surprisingly revealed a mechanism of severing enzyme mediated tubulin exchange along the once-thought static microtubule shaft

Tubulin isoform composition tunes microtubule dynamics

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2Å cryo-EM structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared to brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Lastly, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step towards understanding how tubulin isoform composition tunes microtubule dynamics

Structure and dynamics of single-isoform recombinant Neuronal Human Tubulin

Microtubules are polymers that cycle stochastically between polymerization and depolymerization i.e., they exhibit 'dynamic instability'. This behavior is crucial for cell division, motility and differentiation. While studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogenous, isotypically pure, engineered tubulin. Here we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2 Å structure of unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but is distinguished by subtle differences at polymerization interfaces, which are hotspots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogenous microtubules undergo dynamic instability but they polymerize slower and catastrophe less frequently. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient, but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogenous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well-defined tubulin species and is a first and necessary step towards uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics