Shining Light on Microtubule Nucleation: FRET Measurements With Bayesian Analysis of FLIM

Abstract

The mitotic spindle is a self-organizing structure that segregates chromosomes during cell division. The spindle is composed of microtubules and accessory proteins, and microtubules are composed of the protein tubulin. Understanding microtubule nucleation is critical to understanding the assembly and maintenance of the mitotic spindle. Microtubules are nucleated by accessory proteins called microtubule nucleators, which have been proposed to be activated by microtubules. To test this hypothesis, we measure the activity of nucleators not bound to microtubules by studying the gradient in polymer concentrations leaving the spindle boundary. We combine FRET and microscopy to measure the concentration of polymer in Xenopus egg extracts. To detect FRET, we measure the lifetime of the excited state of the donor fluorophore, a technique commonly referred to as FLIM. We developed a Bayesian framework for analyzing FLIM measurements.