Project Summary/Abstract Two large macromolecular machines, the spindle and kinetochore, coordinate chromosome segregation at cell division. Errors in their function can lead to cancer and birth defects. While we now know nearly all the molecules required for mammalian spindle and kinetochore function, how they collectively give rise to these machines’ emergent physical properties remains poorly understood. Our long-term goal is to uncover the basic physical design principles of robust and accurate mammalian chromosome segregation. How do thousands of nm-scale molecules, pushing and pulling, give rise to the spindle’s m-scale steady-state architecture and mechanics? How do hundreds of kinetochore molecules work together to ‘compute’ attachment signals for decision-making, and to robustly grip microtubules? This knowledge gap persists because we cannot currently reconstitute these machines in vitro, and lack tools in vivo. To close this gap, we need approaches to finely control molecules and directly exert forces inside dividing mammalian cells – which we recently developed. First, we aim to define the mechanisms and functions of the spindle’s emergent architecture and mechanics. (i) Based on our findings, we will test the idea that opposing stresses in the spindle are not required to give it a steady-state structure, but are instead required to give it mechanical and functional stability. Further, we will define in vivo and in vitro how active and passive forces contribute to building a steady-state spindle. (ii) To uncover the function of the spindle’s specific steady-state shape, we developed an optogenetic approach to acutely and locally control spindle architecture. We will use it to test the role of given architectural modules in space and time through mitosis. (iii) To define the mechanisms underlying the steady- state spindle’s mechanical robustness, we will use microneedle manipulation to deform the spindle, which we recently adapted in mammalian cells, and modeling. Second, we aim to define how the kinetochore’s molecules together enable it to ‘compute' attachment information and robustly grip microtubules. (i) Based on our recent finding that only a few bound microtubules are needed for a kinetochore to allow anaphase, we will quantitatively rewire kinetochore composition to test models for the origin of this exquisite microtubule sensitivity. (ii) Using biophysical approaches we developed to remove and exert forces on kinetochores in vivo, we will define the mechanisms giving rise to the kinetochore’s specialized, strong and dynamic grip. Together, this will provide a framework for understanding, targeting, and rewiring the physical processes of chromosome segregation for both basic and therapeutic purposes. This proposal is innovative in that it tests new hypotheses about the connection between molecular and cellular-scale events, and provides new tools for rewiring molecular-scale forces and assemblies and directly probing mechani...