Scaling of Spindle Length and Chromosome Segregation in Drosophila Embryos

Abstract

Scaling is a common phenomenon in biological systems, and the mitotic spindle is a well-known example that demonstrates subcellular scaling. When a spindle is generated in either Xenopus extracts or liquid droplets, the size of the spindle scales with the volume available in a broad regime. While the phenomenon of scaling is well-described, the biological cause and consequence of scaling are poorly investigated. Notably, scaling plays an important role in the correct functioning of cells, but such functions remain inadequately described and studied.The Drosophila melanogaster embryo provides an excellent system for studying cytoskeletal scaling. During the blastoderm stage, nuclei divide without cell growth. In this dissertation, I show that the speed of chromosome separation gradually decreases during the 4 nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in the length of the spindle, thus ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, I found that two processes contribute to controlling the speed at which chromosomes move at mitotic exit: the activity of molecular motors important for microtubule depolymerization and sliding, and the cell cycle oscillator. Specifically, I found that the levels of Klp10A, Klp67A, and Klp59C, three kinesin-like proteins important for microtubule depolymerization, as well as the level of kinesin-5 Klp61F, contribute to setting the speed of chromosome separation. This observation is supported by quantification of microtubule dynamics indicating that poleward flux rate scales with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Therefore, I propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase. As the scaling relationship between chromosome velocity and spindle length remain robust upon genetic manipulation of various motor proteins, I hypothesize that that physical properties of the microtubule contribute to spindle scaling, and the fraction of kinetochore associated microtubules impact chromosome velocity scaling. Using cryo-EM techniques and computational reconstruction, the three-dimensional structure of the mitotic spindle in the Drosophila embryo will be unveiled. The comparison of the spindle ultratructure at different nuclear cycles reveal a new framework for the scaling of mechanical forces with the size of a subcellular structure.