Rebuilding Cytokinesis One Molecule at a Time

Cells are the fundamental units of life. They make up all living things, from bacteria that live in the soil, to archea that give thermal springs their bright colors, to trees and humans. All of these cells share some common functions: they build themselves from basic building blocks, following the instructions of their genetic blueprint, and procreate by growing and dividing. The building blocks must be taken up from the environment and metabolized, and cell division requires the cell to be able to control its own shape. While these basic tasks are shared across the tree of life, different types of organisms have evolved distinct molecular machineries to complete them. In this thesis, we take a close look at animal cells, and ask how they control their shape as they must do in order to move, eat, sense, and divide. In animal cells, shape is controlled by the cytoskeleton, and in particular by the actin cortex. This cortex is a thin layer of actin _laments that sit underneath the plasma membrane, supporting the cell surface. The _laments are highly dynamic: they are constantly growing, shrinking and being remodeled, with well over 100 different proteins regulating their length and architecture. This molecular complexity, combined with the small size and high density of _laments and their rapid remodeling, makes it extremely difficult to disentangle different functions performed by the actin cortex in living cells. To better understand what fundamental principles govern cortex-based shape control of animal cells, we thus pursue a different approach: instead of studying living cells directly, we build minimal versions, so-called ‘synthetic cells’, from the bottom up. In such a bottom-up reconstitution approach, we isolate proteins (for instance actin) from their native environment, purify them, and bring them back together in vitro, following rational design principles. Consequently, we drastically reduce the complexity of the system, giving us a chance to actually understand what is going on. This allows us to test our assumptions about how cellular processes work in vivo, and discover new functions that are normally hidden in the complexity of the living cell. In this thesis, we use bottom-up reconstitution to ask how animal cells control their shape, with the ultimate aim to build a minimal actin-based cell division machinery…