The Drosophila claret segregation protein is a minus-end directed motor molecule

Walker, R. A.; Salmon, Edward D.; Endow, Sharyn A.

doi:10.1038/347780a0

Cited by 346 publications

(269 citation statements)

References 20 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: 70%

“…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: 63%

“…It has been previously shown that Ncd exhibits motile properties in vitro and that in MT gliding assays recombinant Ncd behaves as a nonprocessive minus end-directed MT motor (McDonald et al, 1990;Walker et al, 1990;Foster and Gilbert, 2000). Our study is the first demonstration of the motor activity of Ncd in living cells.…”

Section: Discussionmentioning

confidence: 67%

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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: 70%

supporting

confidence: 63%

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Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Oladipo

Cowan

Rodionov

2007

MBoC

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“…The ncd motor domain used in this study does not induce microtubule movement in an in vitro translocation assay. However, lack of motility may reflect an improper attachment to the glass surface, as has been suggested from results with truncated kinesin constructs (Yang et al, 1990).…”

Section: Concentration Of Nacl (M)mentioning

confidence: 92%

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Shimizu¹,

Sablin²,

Vale³

et al. 1995

Biochemistry

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ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose chromatography, and trace amounts of contaminants were removed by passing through a MonoQ column. The yield was 20 mg from a 500 mL. culture of E. coli. The purified ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275 nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive ATP, 3H at adenine but not 32P at y-phosphate was retained by the protein on gel filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding to the ncd motor domain is tight, although there is an equilibrium between the protein and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the absence of microtubules, but microtubules activated the turnover to a similar extent to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of mantATP increased at 0.005 s-l, which is likely to reflect the release of endogenous ADP and incorporation of mantATP into the protein. The fluorescence intensity of the ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing the mantADP release. The rate was accelerated more than 1000-fold to 3.3 s-l by the presence of saturating microtubules. The profiles of the mantADP release rate and the mantATP turnover rate versus microtubule concentration were nearly identical, except that the maximal rate of mantATP turnover was 30% lower than the maximal mantADP release rate. This result suggests that mantADP release substantially contributes to the overall cycle time. We have also measured the equilibrium dissociation constants for ncd motor domain binding to microtubules in the presence of ADP [&(ADP) = 4-5 pM] and ATP [&(ATP) = 6-7.5 pM] and the apparent halfmaximal microtubule stimulation of ATPase activity (5-7 pM) under an identical condition. The enzymatic and microtubule-binding characteristics of ncd motor domain reported here are similar to those of kinesin. The notable exception, however, is that &(ADP) for kinesin is 2-3-fold larger than &(ATP), which suggests the existence of a weak binding ADP state in the case of kinesin but not in the case of ncd motor domain. Such a difference could be relevant for understanding the opposite polarity of movement of these two microtubule motors.Eukaryotes generate cytoplasmic motility using two types of filaments, actin and microtubules. Actin-based motility is driven by motors that belong to the myosin superfamily

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“…Sideward motion of other motors has been detected via rotations of filaments driven by multiple motors in gliding assays and by off-axis movement of motor-attached microspheres or quantum dots used as tracking probes. Probe and microtubule rotations imply torque generation for all cytoskeletal motors: myosin (11), dynein (9,(12)(13)(14)(15)(16)(17) and kinesin (kinesin-1 monomers (18) and dimers (10), kinesin-2 (19,20), kinesin-5 (21), kinesin-8 (16,22), and kinesin-14 (23)). Occasional directed sideward steps-as suggested for kinesin-8-may explain microtubule rotations of motor-ensemble gliding assays (22) or the spiralling motion of multimotor-coated microspheres around microtubules (20).…”

Section: Introductionmentioning

confidence: 99%

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Bugiel

Böhl

Schäffer

2015

Biophysical Journal

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Molecular motors translocate along cytoskeletal filaments, as in the case of kinesin motors on microtubules. Although conventional kinesin-1 tracks a single microtubule protofilament, other kinesins, akin to dyneins, switch protofilaments. However, the molecular trajectory-whether protofilament switching occurs in a directed or stochastic manner-is unclear. Here, we used high-resolution optical tweezers to track the path of single budding yeast kinesin-8, Kip3, motor proteins. Under applied sideward loads, we found that individual motors stepped sideward in both directions, with and against loads, with a broad distribution in measured step sizes. Interestingly, the force response depended on the direction. Based on a statistical analysis and simulations accounting for the geometry, we propose a diffusive sideward stepping motion of Kip3 on the microtubule lattice, asymmetrically biased by force. This finding is consistent with previous multimotor gliding assays and sheds light on the molecular switching mechanism. For kinesin-8, the diffusive switching mechanism may enable the motor to bypass obstacles and reach the microtubule end for length regulation. For other motors, such a mechanism may have implications for torque generation around the filament axis.

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The Drosophila claret segregation protein is a minus-end directed motor molecule

Cited by 346 publications

References 20 publications

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

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