Abstract:
:Undiluted cytoplasmic extract prepared from unfertilized Xenopus laevis eggs by low-speed centrifugation (CSF extracts) is useful for reconstitution of egg microtubule dynamics and meiosis-II spindle organization, but it suffers limitations for biochemical analysis due to abundant particulates. Here, we describe preparation and the use of fully clarified, undiluted mitotic cytosol derived from CSF extract. Addition of glycogen improves the ability of this cytosol to reconstitute microtubule organization, in part through improved energy metabolism. Using fully clarified, glycogen-supplemented mitotic cytosol, we reconstituted (i) stimulation of microtubule polymerization by Ran.GTP (Groen, Coughlin, & Mitchison, 2011; Ohba, Nakamura, Nishitani, & Nishimoto, 1999) and (ii) self-organization of highly regular bipolar arrays of taxol-stabilized microtubules that we termed "pineapples" (Mitchison, Nguyen, Coughlin, & Groen, 2013). Both systems will be useful for biochemical dissection of spindle assembly mechanisms. We also describe reliable small-scale methods for preparing fluorescent antibody probes that can be used for live imaging in egg extract systems as well as standard immunofluorescence.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Groen AC,Ngyuen PA,Field CM,Ishihara K,Mitchison TJdoi
10.1016/B978-0-12-397924-7.00023-6subject
Has Abstractpub_date
2014-01-01 00:00:00pages
417-33eissn
0076-6879issn
1557-7988pii
B978-0-12-397924-7.00023-6journal_volume
540pub_type
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