Characterizing Molecular Interactions of the N- and C- Terminal Domains of XRHAMM During Early Mitotic Spindle Assembly

Abstract

Proper mitotic cell division is important to ensure that cells divide correctly, where the chromosomes are evenly split between the two resulting daughter cells. Microtubule nucleation and branching microtubule nucleation are steps in ensuring that the mitotic spindle correctly forms, as the mitotic spindle is composed of microtubules and associated proteins. XRHAMM is a microtubule associated protein (MAP) that plays a role in early mitotic spindle assembly. In order to learn more about the role that XRHAMM plays in early mitotic spindle assembly, this paper will analyze the molecular interactions between the N-terminal domain and the C-terminal domain of XRHAMM with various components that are apart of early mitotic spindle assembly. To determine the interactions between the XRHAMM constructs and microtubules, I performed microtubule binding assays in vitro. I found that both the C-terminal domain and the N-terminal domain bind directly to stabilized microtubule seeds. To examine the relationship between the XRHAMM constructs and the MAP TPX2, a protein driving branching MT nucleation, I performed pull down assays. My results are inconclusive as to whether or not the N-terminal and the C-terminal domains of XRHAMM associate with TPX2 in vitro. Altogether, my work sheds light into the specific molecular interactions that the N- and C-terminal domains of XRHAMM have with various components involved in early mitotic spindle assembly.