Investigating the Biochemical Interaction between Eg5 and TPX2 and Its Role in Branching Microtubule Nucleation

Abstract

Cell division is a critical process for the development of cellular organisms. During cell division, a bipolar structure termed the mitotic spindle must be built from microtubule filaments to align and segregate chromosomes. Branching microtubule nucleation is a unique pathway involved in rapid spindle assembly, characterized by the nucleation of microtubules along the sides of pre-existing microtubules. Previous studies show that TPX2, a microtubule-associated protein, is necessary for proper spindle assembly via branching microtubule nucleation, a pathway that generates the majority of microtubules during spindle assembly. During mitosis, TPX2 also interacts with the plus-end-directed kinesin microtubule motor, Eg5, which bundles microtubules and establishes spindle bipolarity. It is unknown whether TPX2-Eg5 binding is necessary for branching microtubule nucleation. Preliminary results show that TPX2 lacking the Eg5-binding domain fails to bind microtubules. Furthermore, a TPX2 construct (TPX2F710AzF) with a point mutation within the Eg5 binding domain is unable to rescue branching microtubule nucleation in Xenopus egg extract depleted of endogenous TPX2. These results led me to hypothesize that Eg5-TPX2 interaction is necessary to induce branching microtubule nucleation. To determine whether Eg5-TPX2 binding is necessary for the microtubule branching pathway, I conducted a branching microtubule nucleation assay in Eg5-depleted Xenopus egg extract. Surprisingly, videos and images taken via Total Internal Reflection Fluorescence Microscopy (TIRFM) displayed branching in Eg5-depleted extract, indicating that Eg5 is not necessary for branching microtubule nucleation. Although Eg5-TPX2 binding is not necessary for the branching pathway, Eg5-TPX2 binding may still alter microtubule dynamics (e.g., polymerization and nucleation). These results help further our understanding of branching microtubule nucleation and spindle assembly during mitosis.