Preface Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a “tug-of-war” between oppositely-directed motors attached to the same cargo. However, although many experimental and modeling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in a variety of systems have found that inhibiting one motor leads to diminished motility in both directions, which is a “paradox of codependence” that challenges it. In an effort to resolve this paradox, three classes of bidirectional transport models, termed microtubule tethering, mechanical activation, and steric disinhibition, are proposed and a general mathematical modeling framework for bidirectional cargo transport is put forward to guide future experiments.

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.DOI: http://dx.doi.org/10.7554/eLife.07403.001

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P i ) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P i state (by adding free P i ) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.single molecule | mechanochemistry | optical tweezers | molecular motors S ince its discovery nearly 30 years ago (1), kinesin-1-the founding member of the kinesin protein superfamily-has emerged as an important model system for studying biological motors (2, 3). During "hand-over-hand" stepping, kinesin dimers alternate between a two-heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one-head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the . A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding....

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.