PROJECT SUMMARY/ABSTRACT Biology and Biochemistry of the Microtubule Cytoskeleton Microtubules are cytoskeletal polymers that are vital for a wide range of cellular functions. They serve as tracks for microtubule-based motor proteins (i.e., kinesin and cytoplasmic dynein), and perform work when their ends attach to subcellular structures such as kinetochores. Most microtubule-based processes rely on assembly of microtubules into higher order structures such as the mitotic spindle. This in turn depends on cellular factors such as microtubule-associated proteins (MAPs) and microtubule motors, which bind microtubules and organize them in three-dimensional space. How MAPs and motors drive assembly of microtubule-based arrays is not fully understood, particularly under conditions of stress. In addition, the complexity of the microtubule system is augmented by post-translational modifications (PTMs) that are “written” onto microtubules; PTMs alter the affinity of MAPs and motors for the microtubule lattice, providing a mechanism to create functionally distinct subsets of microtubules. Our research is focused on understanding how MAPs and motor proteins engage the MT cytoskeleton to orchestrate essential cellular processes. To address this and fill gaps in our knowledge we: (i) Discovered a second pathway that drives assembly of the mitotic spindle, and developed inhibitors against a motor protein that plays a key role in this pathway; (ii) Developed a screening pipeline to identify proteins that bind to microtubules in a manner that depends on tubulin PTMs; and (iii) Identified a mechanism that may explain how tubulin PTMs are written onto the microtubule lattice. The research proposal benefits from our expertise and will combine in vitro reconstitution and cell biology approaches to address long-standing questions: (a) How do MAPs and motor proteins work together to build the mitotic spindle?; (b) What are the functional outputs of tubulin PTMs?; and (c) How are MTs selected for post-translational modification? Our research will provide new insights into fundamental mechanisms that regulate the biological and biochemical properties and functions of the microtubule cytoskeleton and uncover general principles that inform on other cellular processes (e.g., microtubule organization in neurons).