Using Electron Cryotomography and Coarse-Grained Molecular Dynamics to Study Contractile Mechanisms of Eukaryotic Cell Division Machinery

Abstract

Division in eukaryotic cells depends on an actomyosin ring that forms at the midcell and then contracts throughout cytokinesis via interactions between actin filaments and myosin II motor proteins. While the protein players involved are well studied, the ultrastructure of the actomyosin division machinery has been elusive due to its highly dynamic nature, which makes it difficult to preserve without vitrification. Here we use cryo focused ion beam milling and cryosectioning to examine the actomyosin machinery of fission yeast (S. pombe) for the first time in its native state, at different stages of contraction. Due to the machinery's complex and dynamic nature, and because EM images are static, we used structural data from our tomographic reconstructions to inform and guide 3D coarse-grained molecular dynamics simulations of the actomyosin ring contracting. To make our simulations as realistic as possible, for the first time, we have modeled the myosin ATPase cycle step-by-step and a coarse-grained membrane was used to test the ring's ability to constrict the membrane. By simulating all current models (and many variations) of actomyosin architecture and by comparing the results to tomographic data, we propose a working model for the contractile machinery, including how the actin filaments are arranged, how they might be tethered to the membrane and the structure of myosin II in the ring.