The microtubule cytoskeleton provides an intracellular coordinate system and a mechanical scaffold for a multitude of essential cellular functions. The design principles underlying the dynamic organisation and function of the microtubule cytoskeleton are not understood. Using an in vitro reconstitution approach, we will determine the rules that govern which combination of mechano-chemical elements gives rise to specific large-scale organisation of the microtubule cytoskeleton. We will reconstitute the architecture of bipolar spindles that are essential for the segregation of the genetic material during cell division. For the in vitro reconstitutions, we will use candidate proteins suggested to be crucial by the literature and we will identify as yet unknown proteins with critical activities. We will investigate key fundamental questions: In which region of the multidimensional biochemical parameter space is bipolarity encoded, which is essential for successful cell division? What are the molecular mechanisms that determine size scaling of spindles or of spindle substructures? How do chromosomes position themselves correctly within spindles and how are spindles positioned properly within cells? To validate that the answers obtained from our in vitro reconstitutions are also applicable to the cytoskeleton in vivo, the reconstituted systems will be quantitatively compared to living cells at the global and single molecule level. The results of our experiments will develop theoretical models of cytoskeleton architecture and function. The overall goal of the project is to understand at a mechanistic level how the self-organised architecture of the microtubule cytoskeleton, and its collective dynamic and mechanical properties, derive from the complex interplay between its mechano-chemical constituents. This will link the functional properties of a system to the fundamental biochemistry and biophysics of the system’s components.