Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy

Abstract

Mitotic and meiotic spindles consist primarily of microtubules, which originate from centrosomes and within the vicinity of chromatin. Indirect evidence suggested that microtubules also originate throughout the spindle, but the high microtubule density within the spindle precludes the direct observation of this phenomenon. By using meiotic Xenopus laevis egg extract and employing total internal reflection (TIRF) microscopy, microtubule nucleation from preexisting microtubules could be demonstrated and analyzed. Branching microtubule nucleation is an ideal mechanism to assemble and maintain a mitotic spindle, because microtubule numbers are amplified while preserving their polarity. Here, we describe the assays that made these findings possible and the experiments that helped identify the key molecular players involved.

Keywords: Cell division; Cytoskeleton; Meiotic spindle; Microtubule; Microtubule nucleation; Mitotic spindle; TIRF microscopy; Xenopus laevis egg extract.

Fig. 1

Overview of the basic branching MT nucleation assay, which is performed with Xenopus egg extract. Proteins of interest, such as TPX2, can be immunodepleted to assess their phenotype and role. Recombinantly expressed and purified proteins can then be added back to the depleted extract to test whether they complement the depleted function