Bipolarization and poleward flux correlate during xenopus extract spindle assembly
CSF monopole labeled with X-rhodamine tubulin at speckle levels, before computational alignment (4.541Mb)
CSF monopole labeled with X-rhodamine tubulin at speckle levels undergoing spontaneous bipolarization (4.099Mb)
Second example of a CSF monopole labeled with X-rhodamine tubulin at speckle levels undergoing spontaneous bipolarization (6.156Mb)
Control spindle labeled with Alexa488-IgG to TPX2 (C terminal peptide) (upper panel) and X-rhodamine tubulin at speckle levels (lower panel) (7.104Mb)
Mitchison, Timothy J.
Groen, Aaron C.
Desai, Ankur R.
Salmon, Edward D.
Kapoor, Tarun M.
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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.
Author Posting. © American Society for Cell Biology, 2004. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 15 (2004): 5603-5615, doi:10.1091/mbc.E04-05-0440.