XMAP215 & TPX2: Investigating the Role of Spindle Assembly Factors in Microtubule Nucleation

Abstract

During cell division, microtubules are essential to form a large dynamic array called the mitotic spindle which functions to physically segregate the chromosomes. To accomplish these functions, the nucleation and dynamics of microtubules must be carefully regulated in both space and time. Historically, gamma-tubulin has been widely thought of as the universal MT nucleator of the cell. However, gamma-tubulin activity alone cannot fully explain the nucleation potential of the cell, suggesting the presence of unknown components in the nucleation mechanism. This thesis focuses on the structure of two proteins – XMAP215 and TPX2 – which have both been recently demonstrated by our lab to mediate the nucleation of microtubules, primarily through the activity of their C-termini. The structure of the C-terminus of XMAP215 and the site of its interaction with gamma-tubulin is investigated. In these investigations, the TOG5 domain within the C- terminus of XMAP215 is demonstrated to have a key role in the stability of the protein. The structure of the C-terminus of protein TPX2 is also investigated through a series of crystallization trials. Through these trials the ability of the TPX2 C-terminus to drive phase separation is displayed, and guidelines are proposed to circumvent the difficulties posed by phase separation in future experiments. A crystal hit was also obtained during crystallization trials, the identity of which is investigated in X-ray diffraction studies.