ATPase Kinetic Characterization and Single Molecule Behavior of Mutant Human Kinesin Motors Defective in Microtubule-Based Motility

Shimizu, Takashi; Thorn, Kurt S.; Ruby, and Aaron; Vale, Ronald D.

doi:10.1021/bi9928344

Cited by 28 publications

(30 citation statements)

References 43 publications

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“…It carries a missense mutation that changes an R residue to G in loop 11 of the motor head domain of Kif20b. By analogy to similar mutations in other kinesins, this change is likely to disrupt microtubule affinity and motor function (Hirokawa and Noda, 2008;Ebbing et al, 2008;Reid et al, 2002;Shimizu et al, 2000). We intercrossed the two mutant lines and collected 13 transheterozygous embryos.…”

Section: Resultsmentioning

confidence: 99%

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

et al. 2013

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Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.

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Section: Resultsmentioning

confidence: 99%

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

et al. 2013

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“…Basal ATPase Activity-Slow steady-state ATPase rates in the absence of polymerized tubulin were measured using [␥-32 P]ATP (35). NcKin3 (5 M) was incubated in 12A25ϩ buffer (12.5 mM Aces-KOH, 25 mM potassium acetate, 5 mM MgCl 2 , 0.5 M EGTA, pH 6.8) with 1 mM [␥- 32 P]ATP at 22°C.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Mechanistic Basis of the Nonprocessive Kinesin-3 Motor NcKin3

Adio

Bloemink

Hartel

et al. 2006

Journal of Biological Chemistry

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Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 m/s and correlates positively with motor density. The processivity index (k bi,ratio ) of ϳ3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.The kinesin superfamily of proteins contains molecular motors that move along microtubules. Members of the founding class, Kinesin-1 (conventional), move according to the socalled hand-over-hand mechanism that leads to processive motility (1). This mechanism involves two motor heads that alternately bind to the microtubule and produce a stepwise progression of the motor. It implies that the catalytic cycles of the motor heads alternate because the nucleotide state of the catalytic domain determines the microtubule affinity (2). This mechanism is referred to as alternating site catalysis, and has been proven by measuring the consecutive release of ADP from head 1 and 2 of the kinesin dimer (3-5). ADP release experiments show that half of the enzyme-bound ADP is liberated upon interaction with the microtubule and the other half upon addition of ATP. Afterward, the motor continues to perform processive catalytic cycles because of the interlaced interaction of both motor heads. In agreement, the rate of the second ADP release is at least as fast as the stepping rate. The kinetic model of alternating site catalysis is consistent with the hand-overhand mechanism that predicts shifted phases of the kinetic cycles of both motor heads. All Kinesin-1 motors investigated so far conform to this model.Conversely, the nonprocessive Kinesin-14 motor Ncd does not show the ADP release pattern typical for alternating site catalysis. Ncd is a homodimeric kinesin from Drosophila melanogaster and is involved in mitosis and meiosis (6 -8). It moves to the microtubule minus end, and it most likely generates motility because of a molecular power stroke...

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“…Phosphate was separated from nucleotide and protein using charcoal (28). The linear parts of these curves were used to calculate the basal ATP turnover if they contained at least 4 data points in the linear part, and if the double amount of kinesin in the assay led to a curve slope double as steep.…”

Section: Methodsmentioning

confidence: 99%

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Hahlen

Ebbing

Reinders

et al. 2006

Journal of Biological Chemistry

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Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.Kinesin-1 (formerly conventional kinesin) proteins are molecular motors that move along microtubules in a stepwise fashion at the expense of ATP hydrolysis energy. A large body of evidence shows that these motors are dimeric and generate motility by the alternating action of two identical motor domains (1-6). A crucial structural intermediate is a Kinesin-1 molecule that is strongly attached to the filament by one nucleotide-free head, whereas the other one remains in a weakly microtubule-binding ADP state. Upon ATP binding, a very rapid structural change of the neck-linker of the microtubule-bound motor head occurs that allows the partner head to lose its ADP and to bind tightly to the microtubule (7). The neck-linker, a short stretch of ϳ10 amino acid residues that follows immediately C-terminal of the catalytic core motor domain, is thought to exist in at least two different conformations, one that is docked or zippered to the motor core, and a flexible undocked structure (8, 9).The docked neck-linker is visible in some crystal structures (10, 11), and its path along microtubule-bound kinesin has been deduced from cryo-EM (8, 12). According to these data, the neck-linker zippers along a groove in the core motor domain between helix 6 on one end, and loop 10 on the other end where it is held by a meshwork of hydrogen bonds. The nature of the undocked conformation is not clear and might differ in truncated, monomeric constructs (that have been used for many of the structural investigations) and dimeric ones.The conformational change of the neck-linker has been used to explain the mechanism of Kinesin-1 motility. It is thought that necklinker docking allows or causes the second head to bind to a forward microtubule binding site. Interestingly, it is unlikely that the docking process of the neck-linker generates the force exerted by kinesin. The free energy of neck-linker docking is surprisingly low (⌬G ϳϪ1 kJ/mol; Ref. 9), and hardly accounts for the force that can be generated by a single kinesin molecule (5-6 pN, Ref. 13). Estimated from crystal structures, the length of the neck-linker is at least 4 nm. Accordingly, ⌬G will be Ն 12 kJ/mol, which is roughly 10-fold larger than the measured free enthalpy change of neck-l...

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ATPase Kinetic Characterization and Single Molecule Behavior of Mutant Human Kinesin Motors Defective in Microtubule-Based Motility

Cited by 28 publications

References 43 publications

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

Kinetic and Mechanistic Basis of the Nonprocessive Kinesin-3 Motor NcKin3

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

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