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Three-dimensional Model to Understand the Cooperative Transport of Kinesin Molecular Motors in DNA-origami Complexes
Motor proteins are fundamentally important as they are involved in many intercellular activities such as cell division, neuronal transport, muscle contraction, and intra-flagellar transport (IFT). Despite decades of work on motor transport both in vivo and in vitro, mechanistic understanding of motor cooperativity for numerous kinesin motors remains a challenging problem. In the past decade, computer simulations have become an important tool in characterizing such cooperative transport by families of kinesin motors, particularly in conjunction with distinct number of motors attached to the in vitro motor-DNA origami complexes. Here, we present a coarse-grained three-dimensional model that recapitulates cooperative transport by kinesin-2 motors in the motor-DNA origami geometry, and sheds light into the role of landing distributions for the motors, as well as the importance of tracks and roadblocks. In particular, we apply our model to two anterograde IFT motors, (fast) FLA8/8 from Chlamydomonas reinhardtii and (slow) KLP11/20 from C. elegans, and show that FLA8/8 substantially loses its efficiency by nearly a quarter during the co-transport. Our results are generally applicable to motor-DNA origami complexes for other kinesin family motors as well, and we anticipate our model to further uncover mechanistic details about motor coordination during multi-motor transport when used in conjunction with experiments
Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
To
uncover their contrasting mechanisms, antimitotic drugs that
inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays
of kinesin-1 and Eg5 motors in which Eg5 “braking” dominates
motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib)
increased gliding speeds, consistent with inducing a weak-binding
state in Eg5, whereas BRD9876 slowed gliding, consistent with locking
Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated
that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4
nM K<sub>I</sub>. Consistent with its microtubule polymerase activity,
Eg5 was shown to stabilize microtubules against depolymerization.
This stabilization activity was eliminated in monastrol but was enhanced
by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted
spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors
impact spindle assembly and architecture through contrasting mechanisms,
and rigor inhibitors may paradoxically have the capacity to stabilize
microtubule arrays in cells