Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract The long-term goal of our research is to decipher the essential molecular mechanisms underlying successful cell division. Errors in this process have been linked to developmental defects and diseases such as cancer. We now know essentially every protein needed for cell division. However, analyzing the precise molecular mechanisms needed for error-free division has continued to be very challenging for at least three reasons. First, cell division in human cells is a highly dynamic process that can be completed in <1 hour, with key steps such as chromosome-microtubule attachments and nuclear envelop reformation taking only a few minutes. Second, the microtubule-based structures that dynamically self-assemble and function in dividing cells can be ~1000- times larger than their nanometer-sized protein components. Third, this multi-step process depends on several distinct protein-protein interactions that can be transient and mitosis-specific. To address these challenges and fill gaps in our knowledge we have: (i) Discovered and characterized cell-permeable chemical inhibitors of key mechanoenzymes (e.g. AAA, ATPases associated with diverse cell processes). These chemical probes can be used to rapidly (typically, within minutes) inhibit or activate (through relief from inhibition) protein function in dividing human cells. We combine these fast perturbations with state-of-the-art microscopy (e.g. lattice light- sheet microscopy) and quantitative image analysis to dissect mechanisms underlying cell division dynamics in human cells. To identify target-specific phenotypes we carry out parallel inhibitor dose-dependent analyses in matched cell lines that are either inhibitor-sensitive or -resistant. (ii) Deciphered how micrometer-sized features (e.g. microtubule length or overlap length) can effectively be measured by nanometer-sized proteins to generate proportionate outputs (e.g. tags or force). For these studies we have generated a biochemical ‘toolbox’ comprised of recombinant forms of the augmin complex, isotypically-pure human tubulin, key microtubule- associated motor and non-motor proteins and g-TuRC (g -tubulin ring complex), the major microtubule nucleator in human cells. (iii) Developed and applied chemical proteomics approaches to ‘capture’ and profile direct, transient and context-dependent protein-protein interactions in living cells. The research proposed benefits from our expertise and will combine chemical, structural and cell biology approaches to answer long-standing questions, including: (a) What are the functions of different AAA mechanoenzymes during cell division and how are their activities regulated? (b) What is the structural basis of g-TuRC-dependent microtubule nucleation and how does this complex contribute to centrosome-dependent and -independent microtubule formation during mitosis? Our research should provide new insights into fundamental mechanisms, uncover general principles that inform on other cellular processes (e.g. microtubule organization in neurons), and may provide starting points for developing new therapeutics.
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