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Vol. 20, Issue 11, 2766-2773, June 1, 2009
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*Systems Biology Department, Harvard Medical School, Boston, MA 02445; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and Cell Division Group, Marine Biological Laboratory, Woods Hole, MA 02543
Submitted January 14, 2009;
Revised March 13, 2009;
Accepted April 6, 2009
Monitoring Editor: Karsten Weis
Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, 
-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either -tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.
These authors contributed equally to this work.
Address correspondence to: Aaron Groen (aaron_groen{at}student.hms.harvard.edu).
Abbreviations used: HuRP, hepatoma up-regulated protein; MCAK, mitotic centromere-associated kinesin; MT, microtubule; TOG, tumor overexpressed gene; TPX2, targeting protein of Xklp2; -tubulin, gamma tubulin.
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