Investigating Cytoskeletal Motor Mechanisms using DNA Nanotechnology

Abstract

The microtubule cytoskeleton plays a vital role in the spatial-temporal organization of subcellular cargo required to maintain homeostasis and direct cell division. Cytoplasmic dynein and kinesin are opposite-polarity, microtubule-based motors that transport a wide variety of cargo throughout eukaryotic cells. While much is known about the stepping mechanism of kinesin from decades of study, cytoplasmic dynein's size and complexity has limited our understanding of its underlying motor mechanism. Here, a minimal, artificially-dimerized dynein motor was observed with two-color, near-simultaneous, high-precision, single-molecule imaging, which reveals the stepping pattern of each motor domain as dynein moves along the microtubule. Although the stepping behavior appeared highly irregular and erratic, with large variability in step sizes, side stepping behavior, and back stepping behavior, dynein did show evidence of tension-based, coordinated stepping. Furthermore, advances in DNA nanotechnology enabled us to engineer a synthetic motor-cargo system, referred to as a chassis, to investigate how multiple cytoskeletal motors work in teams to produce the myriad of motile behaviors observed in vivo. Specifically, the mechanisms that coordinate motor ensemble behavior was examined using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing control of motor type, number, spacing, and orientation in vitro. Ensembles of 1-7 identical-polarity motors displayed minimal interference with respect to directional velocity, while ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species. This experimental system allowed us to test directly the tug-of-war proposed to occur during dynein's delivery to the microtubule plus-end by the kinesin Kip2. This work led to the mechanistic understanding that Lis1/Pac1, CLIP170/Bik1, and EB1/Bim1 proteins function to enhance kinesin's processivity, allowing it to win a tug-of-war and transport dynein toward the microtubule plus-end. Overall, this work elucidated mechanisms of ensemble motor function and dynein's stepping mechanism in addition to building significant tools to further pave the way for future studies to elucidate how cytoskeletal motors function to organize cellular cargos.