Examining XRHAMM’s Binding Partners and Role in Branching Microtubule Nucleation

Abstract

During mitosis, microtubules make up the structural backbone of the mitotic spindle and provide the machinery to segregate chromosomes. The formation of new microtubules (i.e. microtubule nucleation) can happen at centrosomes, near kinetochores of chromosomes, and from pre-existing microtubules (i.e. branching microtubule nucleation) downstream of RanGTP near chromosomes. Branching microtubule nucleation contributes to a majority of microtubules in the mitotic spindle of many cell types and depends on Ran-regulated spindle assembly factors (SAFs) to recruit the universal microtubule template, gTuRC. XRHAMM (Xenopus laevis analog of human RHAMM) is a microtubule associated protein required for early mitotic spindle assembly implied to interact with TPX2 – an essential Ran-regulated SAF – and gTuRC; however, how XHRAMM contributes to mitotic spindle assembly remains to be uncovered. To start addressing this question, my research is focused on if and how XHRAMM is involved in RanGTP-dependent branching microtubule nucleation. Through pull-down assays, I found full-length XRHAMM directly binds to TPX2. To assess how these proteins could be interacting on the microtubule, I performed sequential microtubule binding assays, but my results are inconclusive as to whether TPX2 is recruiting XRHAMM to the microtubule. Also, through pull-down assays, I found the C-terminal domain of XRHAMM directly binds to gTuRC, whereas the N-terminal domain and full-length XRHAMM do not bind to gTuRC, potentially indicating XRHAMM autoinhibition. These results suggest XRHAMM is part of a TPX2-XRHAMM-NEDD1-gTuRC complex and may have a role in branching microtubule nucleation. Understanding XRHAMM’s binding partners and function is important to explain mitotic spindle assembly, and how overexpression of human RHAMM is connected to various forms of cancer.