Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold

Abstract

Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

Fig. 1

Design and validation of a three-dimensional DNA origami synthetic cargo. (A) Schematic of the twelve-helix bundle chassis structure with 6 inner and 6 outer helices. Each outer helix contains up to 15 optional handles, yielding 90 uniquely addressable sites. Each handle consists of an unpaired 21-bp (~7 nm) oligonucleotide sequence for hybridization to complementary anti-handle sequences covalently attached to motors or fluorophores. Inset shows an orthogonal cross-section. (B) Schematic of a chassis labeled with 5 fluorophores (red) at handle position 14 on each of 5 outer helices and dynein handles at positions 1, 5, 9, and 13 on a single outer helix. Oligonucleotide-labeled dynein is also shown. (C) Agarose gel shift assay of TAMRA-labeled chassis containing 1–4 handles in the absence (left lanes) or presence (right lanes) of dynein labeled with an anti-handle oligonucleotide. Chassis are visualized by TAMRA fluorescence. See fig. S2B for occupancy quantification. (D) Negative-stain TEM images of the 4 dyneinchassis complex. Scale bar, 40 nm.

Fig. 2

Single-molecule motile properties of chassis-motor complexes. (A) Kymographs of TMR-labeled dynein alone and TAMRA-labeled chassis with 1, 2, or 4 dyneins. Plus (+) and minus (−) denote microtubule polarity. Scale bars: 1 min (x), 5 μm (y). (B) Quantification of average segment velocities ± SD of dynein and dynein-chassis complexes. The 4D and 7D ensembles moved significantly slower than dynein alone, or the 1D or 2D ensembles (one-tailed t-test, P < 0.001; N ≥ 211). In higher ionic concentration (↑ ions), the 4D and 7D ensemble velocities were significantly different (one-tailed t-test, P < 0.001; N ≥ 208). (C) Quantification of run lengths ± SE of dynein and dynein-chassis ensembles (N ≥ 208). (D) Quantification of total run times ± SE of dynein and dynein-chassis ensembles (N ≥ 208). (E) Kymographs of TMR-labeled kinesin alone and TAMRA-labeled chassis with 1, 2, or 4 kinesins. Scale bars: 1 min (x), 5 μm (y). (F) Quantification of average segment velocities ± SD of kinesin and kinesinchassis ensembles. Comparison of velocities yielded no statistical differences (ANOVA test, P > 0.05; N ≥ 301). (G) Quantification of run lengths ± SE of kinesin and kinesinchassis ensembles (N ≥ 301). (H) Quantification of total run times ± SE of kinesin and kinesin-chassis ensembles (N ≥ 301). For additional statistical analysis see figs. S4–S6.

Fig. 3

Chassis attached to dynein and kinesin frequently engage in a stalled tug of war. (A) Kymographs of TAMRA-labeled chassis attached to dynein only (left most panel), kinesin only (right most panel), or varying ratios of dynein and kinesin motors (middle panels). Plus (+) and minus (−) denote microtubule polarity. Scale bars: 1 min (x), 5 μm (y). (B) Quantification of the fraction of events for each chassis observed as defined by their dynein to kinesin handle ratio. Chassis were immobile, moving toward the minus end, or moving toward the plus end (table S6, N ≥ 221). X-axis of dynein to kinesin ratios is a logarithmic scale and linear-log fits highlight the trends observed. (C) 10 Quantification of the fraction of events ± SE observed to be immobile, moving toward the minus end, or moving toward the plus end for mixed ensembles containing 2 dyneins and 5 kinesins (N ≥ 352). The dyneins were either wildtype (D) or a highly processive mutant (d^P).

Fig. 4

Disengagement of one motor species resolves stalled tug of war. (A) Schematic of a mixed-motor-chassis with dynein attached via photocleavable handles (purple circles). Photocleavage is induced by 405 nm laser pulses (inset). (B) Kymograph of 2D:5K*(green) and 2D*:5K (red) chassis. Purple lightening bolt indicates the start of laser pulses. Scale bars: 1 min (x), 10 μm (y). (C) Chassis classification scheme for data presented in panel D. Before (pre-state) and after (post-event) laser photocleavage the chassis were characterized as immobile, minus-end-directed, or plus-end-directed. Possible post-events also included dissociation from the microtubule. (D) Quantification of the post-photocleavage event motility of 2D*:5K (top) and 2D:5K* (bottom) chassis as a function of their pre-state (N ≥ 286). Each individual post-event fraction was calculated relative to the number of events within that given pre-state.