Measuring Microtubule Polarity in Spindles with Second-Harmonic-Generation Microscopy

Abstract

Microtubule polarity, the fraction of microtubules that point in one direction versus the other, varies throughout spindles. Near the poles, most microtubules point with their plus ends away from the pole, while in the middle of the spindle, equal number of microtubules point in either direction. Antiparallel microtubules in the spindle midzone are thought to be important for determining the localization of proteins that drive spindle elongation in anaphase. While in vitro experiments have suggested mechanisms connecting antiparallel microtubules, protein localization, and spindle elongation, it has been difficult to establish the validity of these models in vivo. A major challenge is that current methods for measuring microtubule polarity, based on electron microscopy, are extremely labor intensive, challenging to combine with protein localization studies, and only provide static snapshots.Building off of approaches that have been pioneered in neuroscience by Watt Webb's group, we have developed a method using non-linear optical microscopy - second harmonic generation and two-photon fluorescence - to map microtubule polarity throughout spindles. This new technique allows us to quantitatively and nondestructively measure the polarity of microtubules in spindles at high resolution, and study how the distribution of polarity changes over the course of anaphase. We will present a detailed description of the methodology and preliminary results on the temporal evolution of microtubule polarity in the first mitotic spindle of C. elegans.