Characterization of TPX2 in Branching Microtubule Nucleation

Abstract

The process in which a cell parts into two daughter cells, or cell division, is highly dependent on the correct formation of the mitotic spindle, the array of microtubules that pulls the daughter chromosomes apart. Defects in spindle formation may lead to aneuploidy, which destabilizes the genotype of cells and may ultimately lead to malignant, uncontrolled cell growth, or cancer. Hundreds of proteins take part in the formation of the mitotic spindle. One of those molecular players is TPX2. This microtubule-associated protein is essential for branching microtubule nucleation, which may be responsible for the formation of the high microtubule mass within the spindle. Although the occurrence of branching microtubule nucleation is clear, the mechanisms by which it occurs and the role TPX2 plays in it are still unclear. In addition, overexpression of cellular TPX2 levels has been linked to many types of cancer. For this reason, TPX2 is used as a marker for certain types of tumors, suggesting a significant importance of this protein in tumor genesis. It is a very promising anti-cancer drug target, but the lack of molecular understanding of this protein stalls its bright future. This research aimed to clarify the contradicting results in literature regarding TPX2 activities, and to further characterize its role in branching microtubule nucleation. First, a thorough secondary structure analysis was used to define new, conserved alpha-helical regions, which helped design multiple truncated constructs of TPX2. Second, the protein constructs were cloned, expressed in bacteria, purified, and tested for branching microtubule nucleation activity in Xenopus laevis egg extract. This study reports a new minimal region of TPX2 required for successful branching microtubule nucleation to occur. Additional aims were set to determine the microtubule binding and nucleating activity of the conserved regions, and determine their protein interactions. Results from this study will help guide future research on TPX2, and help understand TPX2 as a key molecule in mitosis, to design effective models of branching microtubule nucleation and mitotic spindle formation.