Estimation of microtubule-generated forces using a DNA origami nanospring

Abstract

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.

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

“…Microscopy chambers were prepared using slides and coverslips treated with oxygen plasma and immediately silanized with a mixture of 2% dichlorodimethyl silane and 98% octamethylcyclooctasilane) for 5 min (Gell et al, 2010; Maleki et al, 2022). Before an experiment, antibodies against DIG (Roche 11333089001) or tubulin (TU-20, Abcam) were adsorbed to the silanized glass, followed by a passivation with 1% Pluronic F-127 in MRB80.…”