Chemical Biology of Cell Division - Revision - 2

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Project Summary/Abstract: Our long-term goal is to decipher the molecular mechanisms that ensure the proper assembly and function of the microtubule-based structures needed for error-free cell division. Essentially all the proteins required for cell division in human cells have now been identified. However, uncovering mechanisms has remained challenging as cell division is rapid and can be completed in <1 hour in human cells, with key steps taking only minutes. In addition, the microtubule-based structures needed for cell division require constant energy input to maintain shape and size, cannot be readily isolated in native forms and the protein-protein interactions critical for function can be transient and mitosis-specific. Finally, these structures are micrometer-sized and can be ~1000-times larger than their protein components. We take interdisciplinary approaches that can address these challenges and help dissect the dynamic self-assembly of these essential structures. We have: (i) Discovered and characterized selective cell-permeable chemical inhibitors of key proteins. These inhibitors can be powerful probes to examine cell division dynamics, as proteins can be inhibited or activated (via relief from inhibition), within minutes in living cells. To track the cellular responses to these fast perturbations we use state-of-the-art microscopy (e.g. lattice light-sheet microscopy) and quantitative image analysis methods. (ii) Developed and applied iCLASPI, a chemical proteomics approach to covalently ‘capture’ and profile transient and context-dependent protein-protein interactions in living cells. (iii) Analyzed the self-assembly of basic structural and functional motifs (e.g. bipolar microtubule arrays) with purified proteins. For these biochemical studies, we have assembled a ‘toolbox’ of recombinant proteins including isotypically-pure human tubulin, the augmin complex and key microtubule- associated motor and non-motor proteins. The proposed research, which benefits from our experience and expertise, will focus on anaphase and cytokinesis. These final stages of cell division can be difficult to study using approaches that cannot profile transient protein-protein interactions or do not provide precise temporal control over protein function. We will take interdisciplinary approaches to address the following gaps in our knowledge: (1) What are the roles of microtubule-severing proteins during the final stages of cell division? (2) How do PRC1, a non-motor protein that selectively crosslinks antiparallel microtubules, and kinesin-4, a microtubule plus-end directed motor protein, contribute to the assembly of the spindle midzone during anaphase? (3) What are the minimum number of proteins needed for microtubule-dependent microtubule formation, a key centrosome-independent microtubule nucleation pathway needed for cell division? Errors in cell division have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies commonly used to treat diseases, such as cancer. Our work will not only shed light on fundamental cellular mechanisms but will also catalyze the development of new therapeutics.