Glycogen-supplemented mitotic cytosol for analyzing Xenopus egg microtubule organization.

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 Enzymol

journal_title

Methods in enzymology

authors

Groen AC,Ngyuen PA,Field CM,Ishihara K,Mitchison TJ

doi

10.1016/B978-0-12-397924-7.00023-6

subject

Has Abstract

pub_date

2014-01-01 00:00:00

pages

417-33

eissn

0076-6879

issn

1557-7988

pii

B978-0-12-397924-7.00023-6

journal_volume

540

pub_type

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