The <scp>NLS3</scp> Motif in <scp>TPX2</scp> Regulates Spindle Architecture in <i>Xenopus</i> Egg Extracts

A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet both spindle size and architecture vary dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we characterize a conserved nuclear localization sequence (NLS) motif, ¹²³KKLK¹²⁶ in Xenopus laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA stimulated spontaneous formation of microtubule asters and increased recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles while still binding to the regulatory transport factor importin α. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.