The mitotic spindle is a microtubule-based machine that segregates chromosomes into two new daughter cells when cells divide. Accurate spindle function is critical: mistakes lead to extra or missing chromosomes, which are associated with cancer, birth defects, and miscarriage. Spindle function requires robust coupling of biochemistry and mechanics. Yet, understanding how this self-organizing machine generates the required forces in the right place at the right time remains a challenge. Our long term goal is to determine how micron-scale mechanical properties of the spindle emerge from molecular-scale biochemistry. We focus on microtubule bundles, which provide organization and underpin rigidity in the spindle and in other microtubule-based structures. We do not understand what material properties bundling molecules impart to spindle bundles, how their molecular properties allow them to do so, or how these emergent mechanical properties are tuned for biological functions. To address these questions, we will measure quantitative readouts of how bundles respond to perturbations that alter mechanics. We take a multi-system approach to understanding bundle mechanics in mammalian kinetochore-fibers (k-fibers), which attach and segregate chromosomes; in fission yeast S. pombe spindles, whose stereotyped organization facilitates probing how specific crosslinker properties affect bundles overall; and in vitro, where we have more precise control. Our approach is organized into two programs: (1) probing the molecular and mechanical organization of spindle microtubule bundles, and (2) controlling spindle microtubule bundles to alter function through novel mechanics. In Program 1, we will determine how k-fiber organization balances competing mechanical constraints of robust force-transmission for chromosome segregation with flexibility to adapt to changing spindle morphology. We will also develop new tools to measure force between microtubules within spindle bundles, determining how these bundles effectively transmit force to achieve their mechanical functions. In Program 2, we will determine how the geometric and mechanical properties of microtubule crosslinkers impart bundle-scale properties that are adapted to particular functions. We will create engineered crosslinkers whose mechanical and geometric properties we will control, and use them to build reconstituted microtubule bundles in vitro, and to alter bundle properties in vivo. By measuring the response of these bundles to molecular-scale changes, we will determine how micron-scale properties emerge. In sum, the proposed work will map how molecular scale parts impart spindle bundles with properties that balance competing mechanical constraints. In the long term, this approach may lead to new insight into how altering the cell?s ?building code? can be harnessed to target microtubule architectures with key roles in disease, or to build novel architectures. This approach can extend to understand the emergent mechanics of microtubule bundle architectures beyond the spindle, such as in cilia and axons.
Mistakes in cell division result in cells having extra or missing genetic information, a state which is associated with cancer, miscarriage, and birth defects. The results of this study will provide insight on how components of the machine that segregates genetic information are built, and how their mechanical properties are tuned to ensure that genetic information is segregated accurately when cells divide. In the long run, this approach will potentially suggest new explanations for how disease states related to cell division arise, and new strategies for targeting them.
