Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.
We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.
Mitchison
T J
TJ
Cell Division Group, Marine Biological Laboratory, Woods Hole, MA 02543, USA. timothy_mitchison@hms.harvard.edu
Maddox
P
P
Groen
A
A
Cameron
L
L
Perlman
Z
Z
Ohi
R
R
Desai
A
A
Salmon
E D
ED
Kapoor
T M
TM
D000818:Animals; D018797:Cell Cycle Proteins; D002457:Cell Extracts / Q000737:chemistry*; D016764:Cell Polarity; D002843:Chromatin / Q000378:metabolism; D005260:Female; D008297:Male; D008540:Meiosis; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins; D015398:Signal Transduction; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C486551:TPX2 protein, Xenopus; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein