Microtubules are cytoskeletal polymers assembled from α and β tubulin subunits that function in essentially all cellular activities. Microtubules can act as “tracks” for intracellular cargo transport, are required for cilia- and flagella-based motility, and establish cell morphology in specialized cells such as neurons. In dividing cells, a bipolar spindle assembles from microtubules and partitions genetic material into two daughter cells. Proper microtubule function in these diverse contexts depends on the assembly dynamics of microtubules and their organization into specialized arrays. Both intrinsic factors including the tubulin isotype composition of microtubules and extrinsic factors including microtubule-associated proteins (MAPs) can impact microtubule assembly dynamics. However, the contribution of tubulin isotype composition to microtubule dynamics is not well understood. In the first part of this thesis, I explore the impact of specific β tubulin isotypes on microtubule dynamics. Microtubules undergo dynamic instability, an intrinsic property in which filaments in bulk equilibrium switch between periods of growth and shrinkage. The rate of polymerization and depolymerization can be quantified, as well as the frequency of switching between these states. I selected the two major β tubulin isotypes (βIIB and βIII) expressed in the vertebrate brain for examination. Using an expression and purification system developed in our lab, I generated recombinant tubulin heterodimers that were isotypically pure in β tubulin composition. I used in vitro reconstitution and total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of individual microtubules assembled from these distinct heterodimers. I found that microtubules assembled with βIIB are substantially more stable, switching from a state of growth to a state of shrinkage (termed catastrophe) three-fold less frequently than their βIII-containing counterparts. These two isotypes differ substantially in the C-terminal tail, a region thought important for modulating interactions with MAPs but whose contribution to microtubule dynamics is not well understood. I found that swapping the C-terminal tails did not substantially alter dynamic instability parameters. These data reveal that isotype-specific polymerization properties are mediated by residue changes in the structured “core” of tubulin, rather than the divergent C-terminal tail. In the second part of the thesis, I examine the contribution of microtubule bundles to chromosome movement during anaphase. As sister chromosomes separate, a specialized array of microtubules called the spindle midzone assembles between the segregating chromosomes. Within this structure, microtubules overlap in the antiparallel orientation and are cross-linked by the non-motor MAP, Protein Regulator of Cytokinesis 1 (PRC1), forming bundles. Current models suggest that the spindle midzone can function to facilitate or restrict chromosome movement, however it is unclear how the accumulation of PRC1 on midzone microtubule bundles impact these activities. Using lattice light sheet microscopy, I examined the time-dependent changes in microtubule overlap length that accompany anaphase chromosome movement. I then selectively disrupted midzone formation by knocking down PRC1 and found that chromosome segregation distance and speed increased. These data support a model in which the spindle midzone, rather than aiding in chromosome segregation, instead restricts chromosome movement. Replacing endogenous PRC1 with a mutant that has reduced microtubule affinity reveals that the change in microtubule overlap length is coupled to the braking function of the midzone. My PhD work provides insight into two areas of microtubule assembly regulation. The studies detailed in chapters 2 and 3 reveal how changes in tubulin primary sequence impact polymerization properties of microtubules in vitro. The studies detailed in chapter 4 reveal how changes in the organization of microtubules in cells contributes to spindle function and chromosome segregation during anaphase
