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Updated: Jul 19, 2026
Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs
Published on: June 27, 2013
Regulation of Op18 during spindle assembly in Xenopus egg extracts
P P Budde1, A Kumagai, W G Dunphy
1Department of Molecular & Cell Biology, University of California, Berkeley, 94720, USA.
Abstract:
Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.
Insights
Oncoprotein 18 (Op18) regulates microtubule dynamics. The polo-like kinase Plx1 promotes spindle assembly by inhibiting Op18 phosphorylation, stabilizing microtubules.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oncoprotein 18 (Op18) is a key microtubule-destabilizing protein.
- Op18 activity is regulated by phosphorylation.
- Understanding Op18 regulation is crucial for cell division.
Purpose of the Study:
- To investigate the role of Op18 phosphorylation sites in Xenopus spindle assembly.
- To identify chromatin-associated regulators of Op18.
- To elucidate the mechanism by which Plx1 affects Op18 and spindle formation.
Main Methods:
- Site-directed mutagenesis of Op18 (Op18-AAA, Op18-EEE).
- Xenopus egg extract assays to monitor spindle assembly.
- Affinity chromatography to assess protein binding.
- Chromatin pulldown assays and kinase identification.
- Plx1 depletion experiments.
Main Results:
- Op18 phosphorylation site mutations differentially affected microtubule stability.
- Op18's microtubule-destabilizing activity correlated with tubulin binding.
- Xenopus polo-like kinase 1 (Plx1) was identified as a chromatin-associated kinase regulating Op18 phosphorylation.
- Plx1 depletion inhibited Op18 hyperphosphorylation and spindle assembly.
Conclusions:
- Plx1 plays a critical role in regulating Op18 phosphorylation.
- Plx1 promotes microtubule stabilization and spindle assembly by inhibiting Op18.
- This study identifies a novel regulatory pathway for microtubule dynamics during cell division.
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