This thesis presents the use of DNA origami nanosprings to investigate the mechanical basis of chromosome segregation, focusing on microtubule-kinetochore (Ndc80) force transmission.

Traditional force measurement techniques lack control over protein interactions. The nanosprings were developed as accessible force sensors and validated against optical trapping, successfully measuring piconewton forces from growing and shrinking microtubules.

A key finding is the stoichiometric control of kinetochore function: by varying the number of atruncated version Ndc80 (jubaea) on the nanosprings, the study demonstrated that microtubule stability and force capture are critically tuned by protein copy number, with higher valency (15-20 copies) leading to significant microtubule stabilization and rescue, mirroring native kinetochore behavior.

The work establishes the DNA nanospring as a powerful tool to dissect mitotic mechanics, revealing that cellular mitotic fidelity depends fundamentally on dynamic protein stoichiometry. It also lays the foundation for reconstituting minimal mitotic spindle systems.

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation.