XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

Abstract

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ⌬EB1 or ⌬XMAP215 spindles leads to faulty kinetochore-microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215-EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215-EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts. INTRODUCTIONMeiotic and mitotic spindles are microtubule (MT)-based structures that segregate chromosomes during cell division (Karsenti and Vernos, 2001;Scholey et al., 2003;Kwon and Scholey, 2004). The architecture of the spindle is established and maintained by the action of molecular motors and microtubule-associated proteins (MAPs) that organize MTs and regulate their polymerization dynamics (Wittmann et al., 2001;Howard and Hyman, 2007). Interestingly, despite fast turnover of spindle MTs by poleward translocation and dynamic instability of plus ends, the overall length and shape of the spindle are maintained throughout metaphase. Although it is accepted that a balance of MT-stabilizing and -destabilizing activities governs spindle length, it is still not understood how distinct MAPs act collectively to regulate MT dynamic instability (Tournebize et al., 2000;Goshima et al., 2005).XMAP215 is the founding member of the XMAP215/ Dis1/Tog protein family that is conserved from yeast to humans. It increases ϳ7-to 10-fold the MT growth rate in vitro by acting as a processive tubulin polymerase, which while residing on MT ends, supports multiple rounds of addition of individual tubulin dimers (Vasquez et al., 1999;Brouhard et al., 2008). In accordance with the role of XMAP215 as an MT growth promoter, depletion of Dis1/ XMAP215/Tog proteins leads to shorter spindles in Xenopus egg extracts and Drosophila S2 cells as well as to defects in spindle morphology in HeLa cells, Schizosaccharomyces pombe, and Caenorhabditis elegans (Matthews et al., 1998;Tournebize et al., 2000;Garcia et al., 2001;Cassimeris and Morabito, 2004;Goshim...

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

“…The concentration of EB1 in vertebrate cytoplasm has previously been reported as ranging between 0.25 and 2 µM in Xenopus egg extract [Tirnauer et al, 2002; Kronja et al, 2009] and tens of nanomolar in S. pombe [Katsuki et al, 2009]. We estimated the concentration of EB1 in HeLa cells to be ~0.25 µM using quantitative western blots (Table I and Supporting Information Figs.…”

Molecular Insights into Mammalian End-binding Protein Heterodimerization

“…Differences or changes in the dimerization properties of the three EBs may, therefore, contribute to the differences in the anti-catastrophe activity. Interestingly, C-terminal EB dimerization domains act as dominant negative mutants and have been used in an array of cellular studies as tools to interfere with the activity of endogenous EB proteins (9,20,(25)(26)(27)(28)(29)(30)(31)(32). They were found to affect all EB species by removing them from microtubule plus ends, which results in an increase in the catastrophe frequency (9).…”

“…Transient overexpression of EB1 or EB3 C-terminal domains has been used as a tool in many studies with the intent to interfere with the binding of ϩTIPs with endogenous full-length EBs in different cell types (9,20,(25)(26)(27)(28)(29)(30)(31)(32). We recently showed that a very similar dominant negative effect is also obtained with an EB1 C-terminal domain lacking the tail region, which is essential for partner binding (9), indicating that sequestration of ϩTIP binding partners is not the prevailing mode of interference.…”

Building the Microtubule Cytoskeleton Piece by Piece

“…In Xenopus egg extracts, an interaction between EB1 and XMAP215 is important for physiological MT growth rates between 10 and 20 m/min, proper spindle assembly, and chromosome segregation (58). This effect was investigated in vitro, where EB1 and XMAP215 combined had a much larger effect on MT growth rates than XMAP215 (10-fold increase) or EB1 (1.5-fold increase) alone with rates up to 20 m/min, the highest rates ever observed outside cells (Fig.…”