Dissecting Protein Complexes in Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts.

Abstract

The mitotic spindle is the microtubule-based apparatus that reliably segregates chromosomes during cell division. Recently, it was discovered that microtubules originate within the mitotic spindle by nucleating off of existing spindle microtubules. This mechanism, termed branching microtubule nucleation, allows the efficient amplification of microtubules while preserving their original polarity as required in the spindle. Three molecular players are known to be involved in this process, namely, the protein TPX2, the protein complex augmin, and the gamma-tubulin ring complex; however, little is known about the assembly of the protein complexes. Here, we use the eight-subunit augmin complex as an example of how to dissect the function and assembly of a protein complex using meiotic Xenopus egg extracts. Specifically, immunodepletion combined with total internal reflection fluorescence (TIRF) microscopy is used to identify the role of the protein complex. In parallel, immunoprecipitation (IP) and tandem mass spectrometry (MS/MS) are used to infer how it is assembled. This approach can be applied to investigate the assembly of other multisubunit protein complexes that function in branching microtubule nucleation and mitotic spindle assembly.