Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly.
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, gamma-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 gamma-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.
Systems Biology Department, Harvard Medical School, Boston, MA 02445, USA. aaron_groen@student.hms.harvard.edu
Aaron C
AC
Groen
Thomas J
TJ
Maresca
Jesse C
JC
Gatlin
Edward D
ED
Salmon
Timothy J
TJ
Mitchison
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
C486738:CKAP5 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C486551:TPX2 protein, Xenopus; C122167:TPX2 protein, human; D014404:Tubulin; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D016547:Kinesins