Motility of Dimeric Ncd on a Metal-Chelating Surfactant:  Evidence That Ncd Is Not Processive

deCastro, Michael J.; Ho, Chih Hu; Stewart, Russell J.

doi:10.1021/bi9829175

Cited by 64 publications

(56 citation statements)

References 21 publications

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“…The rate of MT movement calculated by dividing the width of the bleached zone by the time of the linear increase of the fluorescence in the bleached zone was 0.105 Ϯ 0.001 m/s. The numbers obtained by two independent experimental approaches were consistent with each other and with the velocity of Ncd-based MT movement in vitro determined using a MT gliding assay (0.06 -0.16 m/s (McDonald et al, 1990;Walker et al, 1990;deCastro et al, 1999;Tao et al, 2006). These results strongly suggest that MT movement seen in GFP-Ncd-overexpressing cells is indeed generated by the motor activity of Ncd.…”

Section: Resultssupporting

confidence: 81%

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Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Oladipo

Cowan

Rodionov

2007

MBoC

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The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFPtransfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 m/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis. INTRODUCTIONThe mitotic spindle is a molecular machine that serves for equal segregation of chromosomes during mitosis. The principal structural components of the mitotic spindle are cytoplasmic microtubules (MTs) organized into two polarized (minus ends at the center) radial arrays, located at a distance from each other. MT organization in the mitotic spindle is achieved by MT motors, kinesins and dyneins, which generate force for movement toward the plus or minus ends of MTs and that participate in the transport of chromosomes to the spindle poles, spindle elongation during anaphase, regulation of the MT turnover rates, and spindle morphogenesis (for review, see Sharp et al., 2000b;Karsenti and Vernos, 2001;McIntosh et al., 2002;Scholey et al., 2003;Gadde and Heald, 2004;Wadsworth and Khodjakov, 2004).A special role in the formation and maintenance of the mitotic spindle belongs to the minus-end-directed members of the kinesin-14 subfamily. Unlike conventional kinesin, kinesin-14 family members have motor domains at the carboxy terminus (for review, see Ovechkina and Wordeman, 2003). The best studied kinesin-14 members are Drosophila Ncd (Komma et al., 1991;Matthies et al., 1996) and Saccharomyces cerevisiae Kar3 (Meluh and Rose, 1990). These MT motors, and their mammalian (Matuliene et al., 1999;Mountain et al., 1999;Zhu et al., 2005) and plant (Vanstraelen et al., 2006) homologues, play essential roles in mitosis and meiosis. Their activities are important for the formation of the mitotic spindle poles (Goshima and Vale, 2003; MoralesMulia and Scholey, 2005;Zhu et al., 2005) and for regulation of the distance...

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Section: Resultssupporting

confidence: 81%

“…Such movement has never been observed in control cells (data not shown). Because MT movement continued in the presence of Taxol, we conclude that MT movement cannot be explained by forces Walker et al, 1990;deCastro et al, 1999;Tao et al, 2006). These results strongly suggest that MT A. Oladipo et al…”

supporting

confidence: 50%

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Oladipo

Cowan

Rodionov

2007

MBoC

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“…The extended model fits the non-Michaelis-Menten shape of the velocity curve even better, suggesting that friction might be contributing to the effect. Unlike the present experiments a previous report by deCastro et al (55) shows MT velocities that follow Michaelis-Menten dependence. We do not have an explanation for this discrepancy, although a different fraction of inactive motors could play a role.…”

Section: Discussioncontrasting

confidence: 85%

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

Nitzsche

Dudek

Hajdo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]-and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.kinesin | microtubule | gliding motility assay | fluorescence interference contrast microscopy | mathematical modeling S ingle-molecule techniques, in particular single-molecule fluorescence (1) and optical tweezers (2), have greatly advanced our understanding of motor proteins. Such experiments normally rely on the processivity of the motor, which makes it possible to observe multiple consecutive working cycles (i.e., steps). Although single-molecule mechanical measurements on nonprocessive motors can be performed with optical tweezers (3, 4), they require a sophisticated statistical analysis (5, 6) and, if the motor is to be studied under load, advanced feedback (7) or force-clamp mechanisms (8). The minus-end directed C-terminal kinesin-14, ncd, is such a nonprocessive motor. It has been shown to play an essential role in spindle focusing (9-11) and to slide and cross-link microtubules (12, 13).For the working stroke of a single ncd motor a lever arm model has been suggested (14, 15). However, as reviewed in ref. 16 the triggering event for the working stroke is still under debate. On the one hand, it has been suggested that the working stroke is connected to the ADP release (14, 17). On the other hand, it has been proposed to be coupled to the binding of ATP to the microtubule (MT)-ncd complex (4,15,18). In another study, a twostage displacement of the ncd stalk has been proposed where stalk displacement is initiated by binding of ncd to the MT and completed upon ATP binding (19). However, even though a variety of approaches including X-ray crystallography (15), EM (20-23), optical trapping (4, 24), FRET (19), and computational modeling (25) have been used to study the coupling between nucleotide states and mechanical states of ncd, the exact mechanism remains to be elucidated.Alternatively, nonprocessive motors can be studied in gliding motility assays (26) albeit with the disadvantage that single-motor behavior is difficult to access. On an ncd...

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“…The ATPase Pathway-Scheme 1 shows our model for the Mt⅐Ncd ATPase based on the equilibrium binding studies (33), the pre-steady-state kinetics (14,15,34), the motility (13,17,24), and structural results for Ncd (16 -21, 39, 40). The cycle begins at the star (૽) intermediate, and the experimentally determined rate constants are designated.…”

Section: Discussionmentioning

confidence: 99%

“…First, Ncd translocates to the minus-end of microtubules, while kinesin moves to the plus-end (2,4,11). Additionally, kinesin is a highly processive motor, taking several steps per encounter with the microtubule, while Ncd has been shown to be nonprocessive, both mechanically and chemically (12)(13)(14)(15). Ncd and kinesin also differ in their motility and ATPase rates; Ncd is a much slower motor.…”

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confidence: 99%

A Mechanistic Model for Ncd Directionality

Foster¹,

Mackey

Gilbert

2001

Journal of Biological Chemistry

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Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s ؊1 , yet rate-limiting ADP release from the second motor domain occurs at 1.4 s ؊1 . We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule⅐Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minusend of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor. Ncd1 is a microtubule-activated ATPase of the kinesin superfamily involved in spindle assembly and stabilization during female meiosis and the early divisions of the Drosophila embryo (1-4). Conventional kinesin, the founding member of the kinesin superfamily, moves membranous organelles from the neuron cell body to the synapse (reviewed in Refs. 5-7). The motor domains of Ncd and kinesin are ϳ40% identical in amino acid sequence, and their three-dimensional atomic structures are nearly superimposable (8 -10). Despite this high degree of similarity, there are striking differences in their motor behavior that are important for biological function in vivo. First, Ncd translocates to the minus-end of microtubules, while kinesin moves to the plus-end (2, 4, 11). Additionally, kinesin is a highly processive motor, taking several steps per encounter with the microtubule, while Ncd has been shown to be nonprocessive, both mechanically and chemically (12-15). Ncd and kinesin also differ in their motility and ATPase rates; Ncd is a much slower motor.Comparison of the kinesin and Ncd crystal structures suggests that despite the structural similarity of the individual catalytic motor domains, there is a different overall orientation of the heads within the Ncd dimer in comparison to those of the kinesin dimer, both in solution and when bound to the microtubule (16 -21). The neck linker, a sequence of ϳ15 amino acids directly adjacent to the catalytic core, has been implicated in specifying the direction of movement for kinesin superfamily members and to be important for force generation (17,(22)(23)(24). For Ncd, the neck linker includes 13 amino acids N-terminal to the catalytic core. There is high sequence homology among minus-end directed kinesin motors, yet this neck linker sequence is different fro...

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Motility of Dimeric Ncd on a Metal-Chelating Surfactant: Evidence That Ncd Is Not Processive

Cited by 64 publications

References 21 publications

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

A Mechanistic Model for Ncd Directionality

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