XMAP215-EB1 interaction is required for proper spindle assembly and chromosome segregation in Xenopus egg extract.
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 DeltaEB1 or DeltaXMAP215 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.
Kronja
Iva
I
Department of Cell Biology and Biophysics, European Molecular Biology Laboratory, Heidelberg, Germany.
Kruljac-Letunic
Anamarija
A
Caudron-Herger
Maïwen
M
Bieling
Peter
P
Karsenti
Eric
E
D000818:Animals; D002457:Cell Extracts; D020090:Chromosome Segregation; D005260:Female; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D008941:Spindle Apparatus / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*
C486738:CKAP5 protein, Xenopus; D002457:Cell Extracts; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D029867:Xenopus Proteins