Project Summary Cells perform mechanical tasks across a wide range of processes including segregating chromosomes during cell division. These tasks are accomplished by the organization of force-generating cytoskeletal networks. Micron-scale microtubule networks need both motor and non-motor proteins to move and organize filaments into proper functional mechanical units. Our long-term goal is to decipher the mechanical code that underlies the assembly and function of these networks, using mitosis as a model biological process. To achieve this goal, we will employ biochemical reconstitution, biophysical methods, single-molecule fluorescence microscopy, and live- cell imaging. We will build on our recent publications and unpublished preliminary data to focus on microtubule network mechanics in mitosis in the following three Aims: (1) Determine the mechanical and functional differences between bridging fibers in metaphase and the central spindle microtubule network in anaphase. Specifically, we will dissect the molecular mechanisms of an essential crosslinking non-motor MAP, PRC1, that builds distinct motifs within the mitotic spindle. These features include bridging fibers that connect sister kinetochore fibers in metaphase and the central spindle midzone array in anaphase. PRC1 is cell cycle regulated by CDK/cyclin B, and therefore is a biochemically distinct molecule in metaphase and anaphase. We will assemble and mechanically probe filament networks to understand how the spindle is able to differentially generate forces and remodel itself while moving chromosomes in metaphase and anaphase. Imaging live cells during mitosis that express mutant PRC1 constructs will validate our in vitro findings. (2) Determine the molecular mechanisms for MAP clustering and the functional role of MAP clusters in regulating microtubule organization. Specially, we will examine how intrinsically disordered subdomains within PRC1 contribute to MAP clustering. Our published and preliminary data suggests that PRC1 clusters significantly impede filament sliding, and that the C-terminal unstructured domain mediates this effect. We will employ our biophysical and cell biological tools to determine the effect that reducing clustering has on microtubule organization. (3) Determine how complexes of motor and non-motor MAPs collectively regulate microtubule organization. We will examine how the Kif4A/PRC1 complex generates forces during microtubule sliding, and how a steady-state overlap arrangement produces resistive forces that maintain spindle midzone integrity. Together, our findings should advance our understanding of how micron-scale microtubule networks regulate chromosome motions in mitosis. We aim to elucidate a ‘code’ that defines how the structure and biochemistry of different MAPs gives rise to cellular machinery that can perform mechanical work. Errors in microtubule network assembly due to copy number variations or mutations in essential MAPs are linked to disease...