Ncd Motor Research Articles

Regulation of ncd by the oligomeric state of tubulin

We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335–700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06–0.08 sec −1 and 1.8–2.0 sec −1 for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins.

Visualizing a New Binding Site of ncd-Motor Domain on Tubulin

Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 Å. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both α and β tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.

Kinetics Processivity and the Direction of Motion of Ncd

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209–700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s −1 is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K d values are 1.0 μM for ADP-AlF 4, 1.1 μM for ATP γS, 1.5 μM for ATP, 3 μM for ADP, and 10 μM for ADP-vanadate (in 25 mM NaCl, 22°C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 μM −1 s −1. Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme, {formula not available us MathML} where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k 1 and k −4 are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1–mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1·ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.

N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with </=0.1 mM NEM enhanced MC1 binding to MTs in the presence of MgATP but had no effect on MC1 binding to MTs in the presence of MgAMP-PNP. In comparison, treatment with >/=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.

Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive.

The surface immobilization methods that allowed single-molecule motility experiments with native kinesin have not worked with the ncd motor protein and other kinesin-related motors. To solve this problem, a surfactant (Pluronic F108) was chemically modified with the metal-chelating group nitrilotriacetic acid (NTA) to allow surface immobilization of histidine-tagged microtubule motors. The chelating surfactant provided a convenient and effective method for immobilization and subsequent motility experiments with a dimeric H-tagged ncd protein (H-N195). In experiments with the absorption of H-N195 to polystyrene (PS) beads coated with F108-NTA, a monolayer of H-N195 bound in the presence of Ni2+, while in the absence of Ni2+, the extent of adsorption of H-N195 to PS beads was greatly reduced. In motility experiments with H-N195 immobilized on F108-NTA-coated surfaces, microtubules moved smoothly and consistently at an average speed of 0.16 +/- 0.01 micrometer/s in the presence of Ni2+, while without Ni2+, no microtubules landed on the F108-NTA-coated surfaces. Investigation of H-N195 motility on the F108-NTA surfaces provided several indications that ncd, unlike kinesin, is not processive. First, a critical H-N195 surface density for microtubule motility of approximately 250 molecules/micrometer(2) was observed. Second, microtubule landing rates as a function of H-N195 surface density in the presence of MgATP suggested that several H-N195 molecules must cooperate in microtubule landing. Third, the ATP KM in motility assays (235 microM) was substantially higher than the ATP KM of dimeric ncd in solution (23 microM) [Foster, K. A., Correia, J. J., and Gilbert, S. P. (1998) J. Biol. Chem. 273, 35307-35318].

The centrosome in Drosophila oocyte development

The Drosophila oocyte is a highly specialized cell type whose development utilizes MTOCs in various contexts. Figure 4 (see color insert) summarizes the characteristics of the MTOCs at different stages of oogenesis. Polarized mitoses are required to achieve oocyte determination. In the asymmetric germ-cell divisions that culminate in the egg chamber, the mitotic centrosomes are anchored to the spectrosome or fusome in order to produce the regular branching pattern of the cyst cells. It appears that the primary role of the fusome is to orchestrate the polarity and synchrony of oogenic mitoses. In the absence of fusomes or anchored spindles, the regular interconnected cyst network is lost and the oocyte does not differentiate. It is not known if the spindle itself is asymmetric, or whether either centrosome has equal potential to interact with the fusome. Several models can explain the need for polarized mitoses for oocyte differentiation. In one, an unequal distribution of unknown oocyte differentiation factors occurs from as early as the first cystoblast division. Here, the fusome may be required for the distribution of the factors. In another model, there is a mechanism that measures the number of ring canals in the cell, limiting the choice of oocyte to two potential pro-oocytes. In this model, polarized, synchronous divisions must occur to produce only two cells with the highest number of ring canals. In both of these models the centrosome plays an indirect role. A critical event in the determination of the oocyte is the formation of the MTOC. The oocyte MTOC forms shortly after completion of the germ cell mitoses and establishes a microtubule array along which factors required for oocyte determination are transported. It is unclear how this single MTOC forms in the 16-cell cyst, how the centrosomes become inactivated in the adjoining 15 nurse cells, or why the inactivated centrioles are transported into the oocyte. No molecular components of the MTOC are known except for centrosomin, which accumulates at the MTOC relatively late, at approximately stage 5 or 6 of oogenesis. The MTOC plays a central role in establishing the oocyte's polar coordinates. The oocyte microtubule array is required for the polar localization of axis-determining factors. At midoogenesis the MTOC appears to mediate the reversal of the microtubule array and the migration of the nucleus in the oocyte. The posterior follicle cells signal this reversal after receiving the gurken signal. What changes occur at the MTOC to trigger this cytoskeletal rearrangement? A better understanding of the MTOC's molecular components is necessary before we can begin to unravel the mechanisms underlying these events. The morphology of the MTOC changes after it shifts to the oocyte anterior. Staining with anti-centrosomin antibodies shows that the MTOC changes from discrete nucleus-associated bodies into a broad structure associated with the anterior cortex. The molecular mechanisms underlying this structural rearrangement of the MTOC at midoogenesis are presently unknown. Meiosis I occurs in the absence of centrosomes, but meiosis II spindles are linked by a shared, acentriolar, astral MTOC. The organization of the meiosis I spindle poles requires the NCD motor protein; however, the meiosis I spindle poles are acentriolar and contain no known centrosomal core proteins. The meiosis II astral spindle pole has a unique ring-shaped morphology and contains centrosomal proteins, such as gamma-tubulin. Strong mutations in the maternal gamma Tub37C gene do not block meiosis I, but prevent the progression of meiosis II.

Reversing a ‘backwards’ motor

Ncd, a kinesin-related microtubule motor protein that moves the ‘wrong’ way on microtubules, towards the minus ends, has now been made to move like kinesin, towards plus ends, by fusing regions from outside the kinesin motor domain to the Ncd motor.1,2 Since it is the kinesin motor domain that binds to and moves on the microtubule, the finding that regions outside the motor domain can confer directionality of Ncd movement is remarkable—it implies a structural basis for motor polarity. Here we consider this finding from a structural point of view and discuss the implications for motor function and evolution. BioEssays 20:108–112, 1998. © 1998 John Wiley & Sons, Inc.

Spectroscopic studies of the ncd motor domain.ADP complex: CD spectrum of ADP induced by binding to the motor domain of ncd.

Previously, we reported that the nucleotide-free ncd motor domain exhibited a near-UV CD spectrum different from that of the ordinary ncd motor domain.ADP complex [Shimizu and Morii, (1996) J. Biochem. 120, 1176-1181]. In the present study, we exchanged the bound nucleotide ADP with N6-methylADP (MeADP) which has a UV absorption spectrum different from that of ADP. The resultant ncd motor domain. MeADP complex gave a near-UV CD spectrum different from that of the ordinary ncd motor domain with bound ADP. This result indicates that the bound nucleotide contributes to the near-UV CD spectra to a considerable extent although ADP or MeADP free in solution gives a spectrum with negligible peaks and troughs. In addition, the absorption intensity of ADP or MeADP at the peak wavelengths decreased to a considerable extent upon binding to the nucleotide-free ncd motor domain. It is suggested that interaction between adenine moiety and chromophore(s) of the protein contributed to the spectral changes of ADP. A candidate chromophore is Tyr442 which is stacked with the adenine moiety at a distance of 0.43 nm. On the other hand, we detected an intensity decrease of tryptophanyl fluorescence upon binding of a nucleotide to the nucleotide-free ncd motor domain, while at the same time tyrosyl fluorescence increased. The fluorescence changes, as well as the UV absorption change described above, gave similar rates upon addition of a nucleotide to the nucleotide-free ncd motor domain. Therefore, they are likely to originate from the same conformational change of the protein.

Two-Dimensional Tracking of ncd Motility by Back Focal Plane Interferometry

A technique for detecting the displacement of micron-sized optically trapped probes using far-field interference is introduced, theoretically explained, and used to study the motility of the ncd motor protein. Bead motions in the focal plane relative to the optical trap were detected by measuring laser intensity shifts in the back-focal plane of the microscope condenser by projection on a quadrant diode. This detection method is two-dimensional, largely independent of the position of the trap in the field of view and has ∼10- μs time resolution. The high resolution makes it possible to apply spectral analysis to measure dynamic parameters such as local viscosity and attachment compliance. A simple quantitative theory for back-focal-plane detection was derived that shows that the laser intensity shifts are caused primarily by a far-field interference effect. The theory predicts the detector response to bead displacement, without adjustable parameters, with good accuracy. To demonstrate the potential of the method, the ATP-dependent motility of ncd, a kinesin-related motor protein, was observed with an in vitro bead assay. A fusion protein consisting of truncated ncd (amino acids 195–685) fused with glutathione-S-transferase was adsorbed to silica beads, and the axial and lateral motions of the beads along the microtubule surface were observed with high spatial and temporal resolution. The average axial velocity of the ncd-coated beads was 230 ± 30 nm/s (average ± SD). Spectral analysis of bead motion showed the increase in viscous drag near the surface; we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the nonhydrolyzable nucleotide adenylylimidodiphosphate.

Kinetic Mechanism of Monomeric Non-claret Disjunctional Protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.

Reversal in the direction of movement of a molecular motor.

Kinesin and non-claret disjunctional (ncd) are molecular motors of the kinesin superfamily that move in opposite directions along microtubules. The molecular basis underlying the direction of movement is unclear, although it is thought to be an intrinsic property of the motor domain, a conserved region about 330 amino acids in length. The motor domain is found at the amino terminus in conventional kinesins, but at the carboxy terminus in ncd. Here we report on a chimaera composed of the motor domain of the minus-end-directed kinesin of Neurospora crassa. The bacterially expressed fusion protein was tested in motility assays using polarity-marked microtubules. Surprisingly, the chimaera moved towards the plus end, demonstrating that the polarity of force generation of the ncd motor domain has been reversed. This finding indicates that the domain organization, particularly the position of the motor domain, is of fundamental importance for the polarity of force production. It also demonstrates that the direction of microtubule movement is not controlled solely by the motor domain.

Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins.

The Ncd microtubule motor protein is required for meiotic and early mitotic chromosome distribution in Drosophila. Null mutant females expressing the Ncd motor fused to the Aequorea victoria green fluorescent protein (GFP), regulated by the wild-type ncd promoter, are rescued for chromosome segregation and embryo viability. Analysis of mitosis in live embryos shows cell cycle-dependent localization of Ncd-GFP to centrosomes and spindles. The distribution of Ncd-GFP in spindles during metaphase differs strikingly from that of tubulin: the tubulin staining is excluded by the chromosomes at the metaphase plate; in contrast, Ncd-GFP forms filaments along the spindle microtubules that extend across the chromosomes. The existence of Ncd-GFP fibers that cross the metaphase plate suggests that Ncd interacts functionally with chromosomes in metaphase. Differences are no longer observed in anaphase when the chromosomes have moved off the metaphase plate. A mutant form of Ncd fused to GFP also localizes to spindles in live embryos. Mutant embryos show frequent centrosome and spindle abnormalities, including free centrosomes that dissociate from interphase nuclei, precociously split centrosomes, and spindles with microtubule spurs or bridges to nearby spindles. The precociously split and free centrosomes indicate that the Ncd motor acts in cleavage stage embryos to maintain centrosome integrity and attachment to nuclei. The frequent spindle spurs of mutant embryos are associated with mis-segregating chromosomes that partially detach from the spindle in metaphase, but can be recaptured in early anaphase. This implies that the Ncd motor functions to prevent chromosome loss by maintaining chromosome attachment to the spindle in metaphase, consistent with the Ncd-GFP fibers that across the metaphase plate.

Three-dimensional Structure of ncd-decorated Microtubules Obtained by a Back-projection Method

We have used cryo-electron microscopy and image analysis to obtain the three-dimensional (3D) structure of 11, 12, 14 and 15 protofilament microtubules decorated with the motor domain of ncd. To obtain the 3D maps, we developed a back-projection method that does not require a helical arrangement of the tubulin heterodimers. This method allows the calculation of 3D maps even when lattice discontinuities (seams) are present. The maps show that the microtubules we studied conform to a B-type lattice with one or more seams. In the presence of 5′-adenylim-idodiphosphate (AMP-PNP), the motor domain of ncd binds to the microtubule protofilament crest interacting with only one protofilament. Viewing the structures along the microtubule axis shows that the ncd motor domain and the tubulin are tilted in opposite directions. We determined that a clockwise tilt of the tubulin subunits corresponds to a view from the minus end towards the plus end of the microtubule.

A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity.

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.

Crystal structure of the motor domain of the kinesin-related motor ncd.

Microtubule-based ATPases of the kinesin superfamily provide the motile force for many animated features of living cells. Kinesin motors differ in their direction of movement along microtubules. Kinesin and ncd, a kinesin-related motor involved in formation and maintenance of mitotic and meiotic spindles, move in opposite directions along microtubules, even though their motor domains are 40% identical in amino-acid sequence. Here we report the crystal structure of the MgADP complex of the Drosophila ncd motor domain determined to 2.5A by X-ray crystallography, and compare it to the kinesin structure. The ncd and kinesin motor domains are remarkably similar in structure, and the locations of conserved surface amino acids suggest these motors share a common microtubule-binding site. Moreover, structural and functional comparisons of ncd, kinesin, myosin and G proteins indicate that these NTPases may have a similar strategy of changing conformation between NTP and NDP states. We propose a general model for converting a common gamma-phosphate-sensing mechanism into opposite polarities of movement for kinesin and ncd.

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.

Three-dimensional structure of a tubulin-motor-protein complex.

The kinesin superfamily is a class of microtubule-based mechano-enzymes involved in intracellular transport and chromosome movements. Molecules that move towards either the plus end or the minus end of microtubules are represented within the family. The motor domains of these molecules exhibit considerable sequence homology and contain both the ATP- and microtubule-binding sites (reviewed in refs 1, 2). Here we focus on non-claret disjunctional (ncd), a minus-end-directed motor involved in chromosome segregation in meiosis and early mitosis in Drosophila. We have calculated a three-dimensional map of tubulin sheets decorated with monomeric recombinant ncd motor domains by negative-stain electron microscopy and image analysis. Comparisons with a control structure of tubulin alone reveal that each motor domain binds to the crest of a single protofilament, making extensive contacts with both the alpha and beta tubulin monomers. Binding of the motor domain results in significant conformational changes in both of the tubulin monomers.

Crystallization and preliminary structural studies of the ncd motor domain.

The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 A, b = 122.3 A, c = 68.0 A, and there is one ncd molecule per asymmetric unit. The crystals diffract X-ray to at least 2.3 A and are appropriate for high-resolution structure determination.

Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays

The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis. The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends. The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain. In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region. Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg(2+)-ATPase activity and binds microtubules in pelleting assays. In contrast to near full-length ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays. Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis. The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle. If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.