to bind differing ratios of active and inactive motors
(table S6) moved with reduced velocity (fig. S7),
demonstrating that intermotor negative interfer-
ence decreases cargo velocity.
We next investigated the motility of the chassis linked to mixed ensembles of opposite-polarity
motors. We quantified the motility of the chassis
as a function of the dynein-to-kinesin (D:K) ratio
(table S6). All mixed-motor ensembles moved
unidirectionally (Fig. 3A) with no reversals detected
at a precision of ~10 nm. With the exception of
the 1D:6K chassis, all ensembles were more likely
to move toward the minus end of the microtubules
(Fig. 3B). Mixed-motor ensembles were relatively
insensitive to increasing the number of kinesin
motors compared with increasing the number of
dynein motors, which could be due to kinesin ensembles operating predominantly through the actions of fewer motors at any given time (24).
Based on the stall forces of dynein [~5 pN (25)]
and kinesin [~7 pN (26)], we expected that kinesin
plus-end runs would have been more dominant.
In contrast, our results suggest that stall force was
not the only parameter governing the behavior of
opposite-polarity motor ensembles (27). Other parameters, such as microtubule affinity, detachment force, and velocity-dependent on-rates, could
also be relevant (20–22, 28–31). Mixed-motor
ensembles moved more slowly and for longer periods of time than did equivalent single-motor–
type ensembles (fig. S8, A and B), with the
magnitude of this effect being more pronounced
in the plus-end direction. Notably, mixed ensembles of dynein and kinesin were more likely
to be immobile than identical-motor ensembles,
suggesting that opposite-polarity motors engage in a tug-of-war that prevents cargo movement
Based on the longer run lengths and times of
yeast dynein compared with human kinesin, we
hypothesized that dynein runs dominated in mixed-motor ensembles due to dynein’s higher microtubule affinity. To test this, we purified a mutant
dynein with a higher processivity and affinity for
microtubules (denoted dP) (17) and paired it with
kinesins. The 2dP:5K ensemble was even more
likely to move in the dynein direction and had
fewer immobile chassis structures compared with
the 2D:5K ensemble containing wild-type (WT)
dynein (Fig. 3C). These results suggest that
track affinity is a key motor property in governing
opposite-polarity motor ensemble motility. Mixed
ensembles containing the high-affinity dynein mutant also produced slower plus-end runs and longer
run times in both directions compared with the
equivalent WT system (fig. S8, C and D).
We wanted to determine if mixed-motor en-
sembles were nonmotile due to a stalled tug-of-
war. To regulate motor attachment to the chassis,
we introduced photocleavable linkers in selected
handles such that illumination with a 405-nm laser
released one motor type from the chassis (Fig.
4A). We designed two modified chassis: (i) 2D:5K*,
with photocleavable (*) kinesins, and (ii) 2D*:5K,
with photocleavable dyneins. We monitored the
motile properties of these chassis structures be-
fore and after laser-induced photocleavage (Fig.
4B). Cleavage was rapid (fig. S9); within seconds
of photocleaving motors of one type, immobile
chassis moved in the direction of the remaining
motors (Fig. 4B). We classified the state of each
chassis before and after photocleavage (Fig. 4C)
and found that the majority of stalled tug-of-war
events were resolved into active motility (Fig.
4D), indicating that disengagement of one mo-
tor type can resolve tug-of-war events between
dynein and kinesin. Though we also observed rare
events in which ensembles switched directions af-
ter photocleavage, we more commonly observed
that chassis would dissociate when moving in the
direction of the cleaved motor (fig. S10).
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Acknowledgments: We thank C. Lin for assistance with
electron microscopy; F. Aguet for assistance with data
analysis; J. Huang, W. Qiu, W.B. Redwine, and A. Roberts
for helpful advice and critical reading of the manuscript;
members of the Reck-Peterson and Shih labs for advice
and helpful discussions; and J. Iwasa for illustrations.
EM data were collected at the Center for Nanoscale Systems,
Harvard University. DNA-PAINT data were collected at the
Nikon Imaging Center, Harvard Medical School. R.J. is
supported from the Alexander von Humboldt Foundation
through a Feodor Lynen fellowship. S.L.R.-P. is funded
by the Rita Allen Foundation, the Harvard Armenise
Foundation, and a NIH New Innovator award (1 DP2
OD004268-1). W.M.S. is funded by NIH awards
(1U54GM094608 and 1DP2OD004641) and ONR
awards (N000014091118 and N000141010241).
Materials and Methods
Figs. S1 to S10
Tables S1 to S6
References (39, 40)
caDNAno File of Chassis Structure
29 June 2012; accepted 21 September 2012
Published online 11 October 2012;