Uncovering the emergent mechanics of the mammalian spindle
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Uncovering the emergent mechanics of the mammalian spindle


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.

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