Each time a cell divides, it must accurately and equally separate its chromosomes into the two new daughter cells. The machinery in the cell that segregates chromosomes is called the spindle. To perform this task, the spindle must be dynamic, with its microtubules turning over in the order of seconds, in order to generate and respond to forces. At the same time, it must also maintain its shape and structure throughout this process, which lasts about an hour. How the spindle holds itself together without falling apart, remains poorly understood. Inside the spindle, kinetochore-fibers (k-fibers) connect to and move chromosomes. To accurately dictate chromosome movements in the cell, k-fibers must be robustly anchored and correctly oriented within the spindle. In this work, I ask how k-fibers are anchored within the spindle in space and time. While we know most of the molecules required for mammalian spindle function, how these molecules give rise to robust structure and mechanics remains poorly understood. In large part, this is because 1) we lack perturbative tools to probe the mammalian spindle’s mechanics inside cells, and 2) we lack theoretical models to help determine the molecular-scale forces essential for robust spindle mechanics, connecting nanometer- to micrometer- scales.In my thesis work, I have addressed this gap by developing experimental and modeling tools to study k-fiber anchorage inside cells. First, I developed an experimental perturbation called microneedle manipulation to directly exert spatiotemporally controlled forces on individual k-fibers to mechanically challenge their anchorage to the spindle. I discovered that the k-fiber exhibits spatially distinct mechanical responses upon force application, being able to pivot near poles, but not near chromosomes, indicating the presence of specialized (space) and short-lived (time) reinforcement in the spindle center, mediated by a crosslinking protein PRC1. Such reinforcement could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales. Second, I developed a coarse-grained model of the k-fiber to determine the precise spatial regulation of spindle forces essential for its robust anchorage. I discovered that in addition to end-point anchorage, lateral anchorage along the k-fiber’s length is also required, and that a local distribution within 3 μm of chromosomes is necessary and sufficient to robustly preserve the k-fiber’s orientation in the spindle center while still allowing pivoting at poles. This spatial regulation promotes bi-orientation of chromosomes and microtubule-end clustering at poles, thereby providing structural integrity while also supporting different key functions in different regions across the spindle.

Proteins and ligands sample a conformational ensemble that governs molecular recognition, activity, and dissociation. In structure-based drug design, access to this conformational ensemble is critical to understand the balance between entropy and enthalpy in lead optimization. However, ligand conformational heterogeneity is currently severely underreported in crystal structures in the Protein Data Bank, owing in part to a lack of automated and unbiased procedures to model an ensemble of protein-ligand states into X-ray data. Here, we designed a computational method, qFit-ligand, to automatically resolve conformationally averaged ligand heterogeneity in crystal structures, and applied it to a large set of protein receptor-ligand complexes. In an analysis of the cancer related BRD4 domain, we found that up to 29% of protein crystal structures bound with drug-like molecules present evidence of unmodeled, averaged, relatively iso-energetic conformations in ligand-receptor interactions. In many retrospective cases, these alternate conformations were adventitiously exploited to guide compound design, resulting in improved potency or selectivity. Combining qFit-ligand with high-throughput screening or multi-temperature crystallography could therefore augment the structure-based drug design toolbox.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl₂, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.