Decoration of the microtubule surface by one kinesin head per tubulin heterodimer

136

142

Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single-and double-headed kinesin and double-headed ned. Threedimensional density maps and difference maps show that, in adenosine 5'-[P,y-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ned heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ned points toward the minus end.Kinesin and ncd belong to a family of proteins that utilize the energy of ATP hydrolysis to move on microtubules (mts). Their directions of motility are opposite: kinesin toward the plus end; ncd toward the minus end (1-3). Both are dimeric molecules with two head domains, a coiled coil rod, and a tail that binds to cargo (4-7). The head (motor) domains expressed in bacterial systems are functional in ATPase and mt binding and translocate mts when properly attached to a surface (8-12). Despite their opposite directions of movement, the motor domains of kinesin and ncd have many similarities: they are homologous in amino acid sequence and atomic structure (13,14); monomeric constructs of motor domains bind to mts with a stoichiometry of one head per tubulin dimer (9, 10, 15, 16); they compete for binding to equivalent sites on tubulin dimers (16-18); the three-dimensional (3D) structures of tubulin protofilaments (pfs) decorated with monomer kinesin or ncd heads are indistinguishable at low resolution (18)(19)(20) MATERIALS AND METHODSKinesin, ned, and Microtubules. KA401, KA340 (the first 401 and 340 N-terminal residues of rat kinesin heavy chain) and NA295-700 [the C-terminal domain, residues 295-700 of nonclaret disjunctional (ncd), also known as MC5] were prepared and purified as described (16).To isolate mts with 16 pfs, testes of crickets (Acheta domesticus) were dissected, homogenized by a hand homogenizer, suspended in an extracting solution (5 mM Pipes/0.5 mM EGTA/2 mM MgSO4/1 mM DTT/10 ,uM taxol, pH 7.0), and centrifuged at 150 x g for 10 min. The pellet was resuspended in extracting solution and centrifuged at 1000 rpm for 5 min.The supernatant contained sperm tail axonemes released from their heads. Resuspension and centrifugation were repeated several times, and each supernatant was checked by light microscopy. The fractions with the highest axoneme concentration and lowest background were used. For decoration with NA295-700, ...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.

Hirose

Lockhart

Cross

et al. 1996

136

142

“…Binding of the second head domain to either the a or subunits of tubulin would be prevented if the configuration of the dimer directed the tethered head domain away from the MT. Another possibility is suggested by the selective interaction of kinesin head domains with /3-tubulin (14), the decoration of MTs by kinesin head domains with a spacing of 8 nm that corresponds to the repeat distance of (3-tubulin subunits approximately along a single protofilament, the strong binding of one head domain per tubulin heterodimer (15), and the indication of some preference for movement in steps of 8 nm (16 (10). through only one head domain could arise from the inability of the dimer to span the 8-nm separation between (-tubulin subunits along a protofilament.…”

Section: Methodsmentioning

confidence: 99%

Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.

Hackney

1994

334

291

The N-terminal 392 amino acids of the Drosophia kinesin a subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on asiation with a microtubule, whereas a shorter monomeric construct (desigated DKH340) releases .95% of its one bound ADP on association with a microtubule. This The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…Do similar principles operate at the molecular level? Traffic jams of motor proteins on cytoskeletal filaments have been predicted theoretically (12)(13)(14)(15)(16), and experiments with purified proteins indicate that motor proteins have properties that may predispose them to form traffic jams: In principle, almost all the motor-binding sites on a microtubule can be occupied if the kinesin-1 (17,18), kinesin-3 (15), or kinesin-8 (19) concentration is high enough. Moreover, abrupt local increases in kinesin-1 density on a microtubule have been reported (15), but whether these were associated with an abrupt decrease in speed was not investigated in these experiments.…”

mentioning

confidence: 99%

Molecular crowding creates traffic jams of kinesin motors on microtubules

Leduc

Padberg‐Gehle

Varga

et al. 2012

204

242

Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions densityand bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.