Biophysics of chromosome segregation: New evidence for a pushing body model of chromosome segregation

Abstract

Chromosome segregation is the essential process during cell division. In all eukaryotes, chromosome segregation is carried out by the spindle, which consists of microtubules and associated proteins. Despite being studied for over a century, the manner by which forces are generated by the spindle and move chromosomes remains poorly understood. In the past few decades, a canonical model of chromosome segregation has emerged and posited that chromosome motion in anaphase is the sum of two independent, mechanistically distinct processes. However, spindles which have been studied in detail are often found to exhibit behaviors that deviate from the predictions of this canonical model. This situation reveals the inadequacy of the canonical model as a general basis for explaining chromosome segregation. To dissect the mechanism, understanding the spatial organization of microtubule polarity and its interplay with protein localization are thought to be crucial, but the mechanism remains poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. In this dissertation, we developed a quantitative method to nonperturbatively measure microtubule polarity throughout spindles using a combination of second harmonic generation and two-photon fluorescence. We validated this method using computer simulations and comparison to structural data on spindles with known polarity. We used this method to measure microtubule polarity throughout the first mitotic spindle in C. elegans embryos. We believe that this method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems. Furthermore, we have investigated the mechanism of chromosome segregation in C. elegans mitotic spindles, human mitotic spindles, and C. elegans female meiotic spindles. We found that these spindles all contain microtubules with both ends between segregating sister chromosomes. Even as chromosomes move towards spindle poles, these inter-chromosomal microtubules slide apart at the same speed as chromosomes. Perturbing inter-chromosomal microtubules causes chromosome motion to immediately cease. Our results are inconsistent with the canonical model, and support a pushing body model proposed ~100 years ago, arguing that the extension of the inter-chromosomal array of microtubules is the solely primary driver of chromosome segregation in diverse systems.