FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2025753616> ?p ?o ?g. }

• W2025753616 endingPage "157" @default.
• W2025753616 startingPage "152" @default.
• W2025753616 abstract "Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis [1Endow S.A. Henikoff S. Soler-Niedziela L. Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.Nature. 1990; 345: 81-83Crossref PubMed Scopus (181) Google Scholar, 2McDonald H.B. Stewart R.J. Goldstein L.S. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor.Cell. 1990; 63: 1159-1165Abstract Full Text PDF PubMed Scopus (281) Google Scholar, 3Hatsumi M. Endow S.A. The Drosophila ncd microtubule motor protein is spindle-associated in meiotic and mitotic cells.J. Cell Sci. 1992; 103: 1013-1020Crossref PubMed Google Scholar, 4Matthies H.J. McDonald H.B. Goldstein L.S. Theurkauf W.E. Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.J. Cell Biol. 1996; 134: 455-464Crossref PubMed Scopus (209) Google Scholar, 5Endow S.A. Komma D.J. Spindle dynamics during meiosis in Drosophila oocytes.J. Cell Biol. 1997; 137: 1321-1336Crossref PubMed Scopus (108) Google Scholar, 6Endow S.A. Waligora K.W. Determinants of kinesin motor polarity.Science. 1998; 281: 1200-1202Crossref PubMed Scopus (133) Google Scholar, 7Foster K.A. Mackey A.T. Gilbert S.P. A mechanistic model for Ncd directionality.J. Biol. Chem. 2001; 276: 19259-19266Crossref PubMed Scopus (31) Google Scholar]. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle [8Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. Structural and functional domains of the Drosophila ncd microtubule motor protein.J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar, 9Karabay A. Walker R.A. Identification of microtubule binding sites in the Ncd tail domain.Biochemistry. 1999; 38: 1838-1849Crossref PubMed Scopus (72) Google Scholar, 10Goshima G. Nedelec F. Vale R.D. Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.J. Cell Biol. 2005; 171: 229-240Crossref PubMed Scopus (195) Google Scholar, 11Tao L. Mogilner A. Civelekoglu-Scholey G. Wollman R. Evans J. Stahlberg H. Scholey J.M. A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays.Curr. Biol. 2006; 16: 2293-2302Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Oladipo A. Cowan A. Rodionov V. Microtubule motor Ncd induces sliding of microtubules in vivo.Mol. Biol. Cell. 2007; 18: 3601-3606Crossref PubMed Scopus (25) Google Scholar]. Although a tailless Ncd has been shown to be nonprocessive [13Case R.B. Pierce D.W. Hom-Booher N. Hart C.L. Vale R.D. The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.Cell. 1997; 90: 959-966Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 14Crevel I.M. Lockhart A. Cross R.A. Kinetic evidence for low chemical processivity in ncd and Eg5.J. Mol. Biol. 1997; 273: 160-170Crossref PubMed Scopus (78) Google Scholar, 15Stewart R.J. Semerjian J. Schmidt C.F. Highly processive motility is not a general feature of the kinesins.Eur. Biophys. J. 1998; 27: 353-360Crossref PubMed Scopus (26) Google Scholar, 16deCastro M.J. Ho C.H. Stewart R.J. Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive.Biochemistry. 1999; 38: 5076-5081Crossref PubMed Scopus (59) Google Scholar], the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along “single” microtubules, whereas it exhibited processive movement along “bundled” microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization. Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis [1Endow S.A. Henikoff S. Soler-Niedziela L. Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.Nature. 1990; 345: 81-83Crossref PubMed Scopus (181) Google Scholar, 2McDonald H.B. Stewart R.J. Goldstein L.S. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor.Cell. 1990; 63: 1159-1165Abstract Full Text PDF PubMed Scopus (281) Google Scholar, 3Hatsumi M. Endow S.A. The Drosophila ncd microtubule motor protein is spindle-associated in meiotic and mitotic cells.J. Cell Sci. 1992; 103: 1013-1020Crossref PubMed Google Scholar, 4Matthies H.J. McDonald H.B. Goldstein L.S. Theurkauf W.E. Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.J. Cell Biol. 1996; 134: 455-464Crossref PubMed Scopus (209) Google Scholar, 5Endow S.A. Komma D.J. Spindle dynamics during meiosis in Drosophila oocytes.J. Cell Biol. 1997; 137: 1321-1336Crossref PubMed Scopus (108) Google Scholar, 6Endow S.A. Waligora K.W. Determinants of kinesin motor polarity.Science. 1998; 281: 1200-1202Crossref PubMed Scopus (133) Google Scholar, 7Foster K.A. Mackey A.T. Gilbert S.P. A mechanistic model for Ncd directionality.J. Biol. Chem. 2001; 276: 19259-19266Crossref PubMed Scopus (31) Google Scholar]. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle [8Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. Structural and functional domains of the Drosophila ncd microtubule motor protein.J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar, 9Karabay A. Walker R.A. Identification of microtubule binding sites in the Ncd tail domain.Biochemistry. 1999; 38: 1838-1849Crossref PubMed Scopus (72) Google Scholar, 10Goshima G. Nedelec F. Vale R.D. Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.J. Cell Biol. 2005; 171: 229-240Crossref PubMed Scopus (195) Google Scholar, 11Tao L. Mogilner A. Civelekoglu-Scholey G. Wollman R. Evans J. Stahlberg H. Scholey J.M. A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays.Curr. Biol. 2006; 16: 2293-2302Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Oladipo A. Cowan A. Rodionov V. Microtubule motor Ncd induces sliding of microtubules in vivo.Mol. Biol. Cell. 2007; 18: 3601-3606Crossref PubMed Scopus (25) Google Scholar]. Although a tailless Ncd has been shown to be nonprocessive [13Case R.B. Pierce D.W. Hom-Booher N. Hart C.L. Vale R.D. The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.Cell. 1997; 90: 959-966Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 14Crevel I.M. Lockhart A. Cross R.A. Kinetic evidence for low chemical processivity in ncd and Eg5.J. Mol. Biol. 1997; 273: 160-170Crossref PubMed Scopus (78) Google Scholar, 15Stewart R.J. Semerjian J. Schmidt C.F. Highly processive motility is not a general feature of the kinesins.Eur. Biophys. J. 1998; 27: 353-360Crossref PubMed Scopus (26) Google Scholar, 16deCastro M.J. Ho C.H. Stewart R.J. Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive.Biochemistry. 1999; 38: 5076-5081Crossref PubMed Scopus (59) Google Scholar], the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along “single” microtubules, whereas it exhibited processive movement along “bundled” microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization. To examine the motility of Ncd, we generated an N-terminal truncation series of Ncd fused with GFP (Figure 1A and Figure S1 available online). We first estimated the oligomeric state of all proteins. Sucrose density gradient centrifugation and gel-filtration analysis showed that GFP-Ncd320 and longer constructs form stable dimers in solution, whereas GFP-Ncd333 forms a monomer (Table S1). We further evaluated the oligomeric state of GFP-Ncd26 and GFP-Ncd195 by measuring the fluorescence intensities of GFP spots with a total internal reflection fluorescence (TIRF) microscope (Figure S4). The fluorescence intensities indicate that these spots correspond to single molecules of Ncd dimer even on the MT, where local motor-protein density could be high. The behavior of single molecules of GFP fusion proteins was tested in a motility assay by using a TIRF microscope at an ionic strength of 5 mM potassium acetate (KAc) in the presence of ATP. A nearly full-length construct, GFP-Ncd26, showed processive motility on microtubules (MTs) (Figures 1B and 1C and Movie S1). Moreover, GFP-Ncd195, which contains the whole stalk domain but lacks the tail domain, also showed processive motility, whereas GFP-Ncd222 and shorter constructs did not (Figure 1D). Although the movement of GFP-Ncd26 and GFP-Ncd195 was primarily unidirectional, there were occasional pauses and brief backward movements (Figure 1C). To quantify the bias of the movements, we calculated the instantaneous velocity from the pairwise distance (Figure 1E and Table 1) [17Inoue Y. Iwane A.H. Miyai T. Muto E. Yanagida T. Motility of single one-headed kinesin molecules along microtubules.Biophys. J. 2001; 81: 2838-2850Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar]. We found that GFP-Ncd26 and GFP-Ncd195 had clear directionality, but GFP-Ncd222 and GFP-Ncd236 only had a small or no bias toward the minus end of MTs (p values for the hypothesis that the mean equals zero were 0.74 and 0.0022 for GFP-Ncd222 and GFP-Ncd236, respectively; Student's t test). For GFP-Ncd26, approximately half of the molecules did not move but remained attached to MTs, probably via the ATP-independent MT binding site in the N-terminal tail domain (amino acid residues 83–100 and 115–187) [9Karabay A. Walker R.A. Identification of microtubule binding sites in the Ncd tail domain.Biochemistry. 1999; 38: 1838-1849Crossref PubMed Scopus (72) Google Scholar], whereas shorter constructs rarely showed such behavior. Thus, for GFP-Ncd26, we classified all runs into moving and nonmoving fractions (see Supplemental Experimental Procedures). The classification seemed to work well: 41% of all tracked runs were excluded, but a considerable population remained with near-zero velocity. This population may reflect that GFP-Ncd26 paused during processive movement, which would lead to an underestimation of the velocity. We next quantified the diffusive element by plotting the mean square displacement (MSD) versus time interval [18Okada Y. Hirokawa N. A processive single-headed motor: Kinesin superfamily protein KIF1A.Science. 1999; 283: 1152-1157Crossref PubMed Scopus (335) Google Scholar] (Figure 1G). The quadratic fit [ρ(τ) = 2Dτ+ v2τ2 + C] to MSD plots of GFP-Ncd26 and GFP-Ncd195 gave a diffusion coefficient D that was larger than that of kinesin-GFP and larger than expected for a Poisson stepper with a step size of 8 nm and a mean velocity of 100 nm/s (D = ∼1000 nm2/s) [19Svoboda K. Mitra P.P. Block S.M. Fluctuation analysis of motor protein movement and single enzyme kinetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 11782-11786Crossref PubMed Scopus (262) Google Scholar]. The values are rather consistent with the movement that can be described as a biased Brownian motion along MTs, as previously discussed [18Okada Y. Hirokawa N. A processive single-headed motor: Kinesin superfamily protein KIF1A.Science. 1999; 283: 1152-1157Crossref PubMed Scopus (335) Google Scholar, 20Kwok B.H. Kapitein L.C. Kim J.H. Peterman E.J. Schmidt C.F. Kapoor T.M. Allosteric inhibition of kinesin-5 modulates its processive directional motility.Nat. Chem. Biol. 2006; 2: 480-485Crossref PubMed Scopus (88) Google Scholar]. The fit also gave a drift velocity v (GFP-Ncd26: −47 ± 22 nm/s; GFP-Ncd195: −91 ± 46 nm/s), consistent with the instantaneous velocity. Mean duration and run length were decreased by N-terminal truncation (Table 1, Figure 1F, and Figure S3): The most marked difference was between the processive motility of GFP-Ncd195 and the nonprocessive motility of GFP-Ncd222, though both proteins had similar velocity in multiple-molecule MT-gliding assays. Thus, we will hereafter focus on GFP-Ncd195 and GFP-Ncd222 to identify the exact factor that ensures processive movement.Table 1Summary of Single-Molecule Motility Assays on Single MTs in the Presence of ATPConstructOligomeric StateVelocity (nm/s)Run Length (nm)Duration (s)Diffusion Coefficient (×104 nm2/s)nMT Gliding Velocity (nm/s)GFP-Ncd26Dimer−46 ± 2−540 ± 578.8 ± 0.51.0 ± 0.1151150 ± 3GFP-Ncd195Dimer−97 ± 4−430 ± 403.4 ± 0.62.0 ± 0.3230135 ± 6GFP-Ncd222Dimer−20 ± 11−40 ± 240.57 ± 0.047.1 ± 3.1207136 ± 10GFP-Ncd236Dimer−20 ± 13−52 ± 160.77 ± 0.012.3 ± 0.6219112 ± 4GFP-Ncd195(E6)Dimer−2.1 ± 0.9−95 ± 162.2 ± 0.10.53 ± 0.11205138 ± 4GFP-Ncd195 on dMTDimer−1.5 ± 1.3−15 ± 263.2 ± 0.10.69 ± 0.1083102 ± 6GFP-Ncd195aSingle-molecule motility assays were carried out on a negatively-charged glass surface. Although the coverslips were coated with casein to prevent nonspecific interactions, we wished to foreclose the possibility that GFP-Ncd195 can interact with the glass surface to move processively on MTs. We therefore observed the motility on a hydrophobic silanized surface. The movement was unidirectional and essentially comparable to that on a nonsilanized glass surface.Dimer−72 ± 2−440 ± 104.6 ± 0.1ND107NDKinesin-GFPDimer690 ± 51620 ± 192.8 ± 0.10.48 ± 0.08109760 ± 16Velocities were determined from Gaussian fits to the instantaneous velocities. The minus (−) symbol refers to the polarity of the MT. Errors are given as the standard error of the fitted parameter, except for the standard errors of run length of GFP-Ncd that were calculated from the raw data. n is the number of runs that were scored for each construct from 3–5 different flow chambers. MT gliding velocities were determined from 30–50 MTs. ND, not determined.a Single-molecule motility assays were carried out on a negatively-charged glass surface. Although the coverslips were coated with casein to prevent nonspecific interactions, we wished to foreclose the possibility that GFP-Ncd195 can interact with the glass surface to move processively on MTs. We therefore observed the motility on a hydrophobic silanized surface. The movement was unidirectional and essentially comparable to that on a nonsilanized glass surface. Open table in a new tab Velocities were determined from Gaussian fits to the instantaneous velocities. The minus (−) symbol refers to the polarity of the MT. Errors are given as the standard error of the fitted parameter, except for the standard errors of run length of GFP-Ncd that were calculated from the raw data. n is the number of runs that were scored for each construct from 3–5 different flow chambers. MT gliding velocities were determined from 30–50 MTs. ND, not determined. We predicted that the processive movement is supported by weak electrostatic interactions between GFP-Ncd195 and MTs. To test this idea, we observed the movement of GFP-Ncd195 in varying salt concentrations (Figure 2A). At 55 mM KAc or higher ionic strengths, the movement of GFP-Ncd195 was diffusive and had only a small bias toward the minus end of MTs (p < 0.0001, Student's t test), whereas kinesin-GFP showed processive motility at 105 mM KAc [21Thorn K.S. Ubersax J.A. Vale R.D. Engineering the processive run length of the kinesin motor.J. Cell Biol. 2000; 151: 1093-1100Crossref PubMed Scopus (226) Google Scholar]. The high sensitivity of GFP-Ncd195 to ionic strength suggests that the movement of GFP-Ncd195 is supported by electrostatic interactions. We further predicted that GFP-Ncd195 is tethered via the negatively charged C terminus of tubulin, known as the E-hook. To test this, we repeated the single-molecule motility assays using subtilisin-digested MTs that lack the E-hook (Figure S5). Figure 2B and Table 1 show that GFP-Ncd195 is unable to move processively along the digested MT. The loss of processivity could be due to structural defects in the digested MT, but the smooth movement of kinesin-GFP in our single-molecule motility assay and a recent structural study [22Skiniotis G. Cochran J.C. Muller J. Mandelkow E. Gilbert S.P. Hoenger A. Modulation of kinesin binding by the C-termini of tubulin.EMBO J. 2004; 23: 989-999Crossref PubMed Scopus (76) Google Scholar] do not support this notion. Hence, we conclude that loss of processivity on subtilisin-digested MTs is due to the absence of the E-hook, as previously discussed [23Lakamper S. Meyhofer E. The E-hook of tubulin interacts with kinesin's head to increase processivity and speed.Biophys. J. 2005; 89: 3223-3234Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar]. Considering the marked difference in processivity between GFP-Ncd195 and GFP-Ncd222, the N-terminal first quarter of the stalk domain (F195-G221) of Ncd195 is sufficient to convert the nonprocessive GFP-Ncd222 construct into a processive one. This region is highly basic, containing six lysines and one arginine in 20 amino acids (hereafter referred to as the “K-rich” region, Figure 2C). To test whether this region is the electrostatic partner of the E-hook, we produced a mutant construct, GFP-Ncd195(E6), in which six lysines in the K-rich region were replaced by glutamates. The velocity and run length of this mutant decreased to levels similar to wild-type on subtilisin-digested MTs (Figure 2B and Table 1), suggesting that these residues are responsible for the stability of the interaction with MTs. It is possible that the K-rich region forms a stable coiled-coil structure, as predicted by the amphipathy score [24Ito M. Morii H. Shimizu T. Tanokura M. Coiled coil in the stalk region of ncd motor protein is nonlocally sustained.Biochemistry. 2006; 45: 3315-3324Crossref PubMed Scopus (6) Google Scholar], therefore we cannot completely rule out the possibility that the mutation could break the coiled-coil structure of this region and cause the observed decrease in velocity and run length. However, this does not seem to be the case. It is unlikely that the K-rich region forms a stable coiled-coil structure for two reasons. First, the K-rich region (F195-G221) and the remaining C-terminal part (E222-R346) of the stalk domain belong to different types of heptad frames and have a proline residue at the boundary that could cause a kink in the structure (Figure S2). Second, the synthetic peptide fragment (K188-T275) containing the K-rich region (F195-G221) is unable to form a coiled coil [25Makino T. Morii H. Shimizu T. Arisaka F. Kato Y. Nagata K. Tanokura M. Reversible and irreversible coiled coils in the stalk domain of ncd motor protein.Biochemistry. 2007; 46: 9523-9532Crossref PubMed Scopus (11) Google Scholar]. Therefore, we consider that the positive charge in the K-rich region, not the coiled-coil structure, ensures processive movement by interacting with the negatively charged E-hook of tubulin. We next examined the biochemical properties of GFP-Ncd195 and GFP-Ncd222 by measuring the MT-activated ATPase activity (Figure 2D). GFP-Ncd195 and GFP-Ncd222 had the same kcat value of 1.6 s−1 but had different Km,MT values of 0.22 and 0.44 μM, respectively. When comparing GFP-Ncd195 with GFP-Ncd222, the decreased Km,MT of GFP-Ncd195 is qualitatively consistent with the longer duration observed in the motility assay, but not quantitatively consistent (two-fold and six-fold for the ATPase and motility assay, respectively). We suppose that the Km,MT values would not directly reflect the observed difference in duration for two possible reasons. First, Ncd molecules that move in short periods (up to about 200 ms) are ignored in the motility assay but are likely to substantially contribute to the ATPase rate. Second, the degradation products that contain the motor domain (Figure S1) might reduce the apparent difference in ATPase activity between the two constructs because these truncated products would have higher ATPase activity and affinity for MTs [26Pechatnikova E. Taylor E.W. Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase.J. Biol. Chem. 1997; 272: 30735-30740Crossref PubMed Scopus (33) Google Scholar, 27Pechatnikova E. Taylor E.W. Kinetics processivity and the direction of motion of Ncd.Biophys. J. 1999; 77: 1003-1016Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Thus, although the difference in the ATPase assay was smaller than that observed in the motility assay, the result is consistent with the idea that the N-terminal region of Ncd195 enhances its affinity for MTs. We speculated that the diffusive element of the processive movement would not require ATP hydrolysis. To test this, we carried out motility assays of GFP-Ncd proteins in the presence of saturating ADP. Notably, GFP-Ncd320 (a dimer containing only the neck and motor domains) as well as GFP-Ncd195 and GFP-Ncd222 exhibited nonbiased one-dimensional diffusion along MTs, whereas kinesin-GFP did not move (Figure S6 and Table S2). GFP-Ncd333, a monomer construct, only showed association-dissociation events in short periods, almost within one frame (data not shown). These results suggest that dimeric Ncd head domains alone can diffuse along MTs in the weak binding state. Table S2 also shows that the K-rich region further supports the association with MTs by interacting with the E-hook. Adding 50 mM KAc to the assay buffer virtually inhibited the unidirectional movement of GFP-Ncd195 along single MTs. Unexpectedly, we found that GFP-Ncd195 moved along bundled MTs even at 105 mM KAc (Figures 3A and 3C). The processive movement was observed when MTs were premixed with GFP-Ncd195 to form MT bundles. This processivity, even when electrostatic interactions were weakened at 105 mM KAc, could be explained by an increased interaction with the E-hooks of bundled MTs. To evaluate this hypothesis, we developed an assaying system specifically designed for monitoring the movement of single molecules on parallel MT bundles in which MTs are grown from an axoneme (Figure 3B). The axonemes define the polarity of the MTs that grow preferentially from the plus end of the axonemes and provide a rigid anchor for the MT bundles. We first confirmed that GFP-Ncd195 formed a dimer on MT bundles by measuring the fluorescence intensity (Figure S4). The bulk of GFP spots moved processively toward the axoneme, even at 105 mM KAc (Figure 3, Table 2, and Movie S2). We also used axonemes themselves as a more stable “rail” for the motility assay because they have doublet MTs that could mimic parallel MT bundles. GFP-Ncd moved processively along axonemes even at 105 mM KAc (Movie S3), as observed along MT bundles. MT bundling increases the number of E-hooks available for the tethering interaction and may prevent the complete dissociation of Ncd from MTs and allow effective rebinding to MTs (Figure 4B). These results indicate that GFP-Ncd195 can move processively, at least along parallel MT bundles. Because the tail region of Ncd interacts electrostatically with the MT, it would be plausible to expect that Ncd could move along “antiparallel” MT bundles that also are seen in mitotic spindles. However, further work is required to address this issue.Table 2Effects of Ionic Strength and Microtubule Bundling on MotilityGFP-Ncd195Kinesin-GFPMT OrganizationKAc (mM)Velocity (nm/s)Run Length (nm)Duration (s)Diffusion Coefficient (×104 nm2/s)nVelocity (nm/s)Run Length (nm)Duration (s)Diffusion Coefficient (×104 nm2/s)nSingle MTs5−97 ± 4−430 ± 403.4 ± 0.62.0 ± 0.3230690 ± 51620 ± 192.8 ± 0.10.48 ± 0.0810955−1.5 ± 0.3−77 ± 362.1 ± 0.10.36 ± 0.0979760 ± 471320 ± 72.0 ± 0.10.61 ± 0.2182105−13 ± 7−22 ± 261.4 ± 0.10.84 ± 1.2081800 ± 291370 ± 192.1 ± 0.10.99 ± 0.2645MT Bundles105−84 ± 5−380 ± 222.9 ± 0.10.42 ± 0.06209NDNDNDNDAxonemes5−65 ± 4−440 ± 385.6 ± 0.10.81 ± 0.13108640 ± 41960 ± 302.6 ± 0.10.37 ± 0.084555−74 ± 2−430 ± 354.1 ± 0.10.84 ± 0.13111NDNDNDND105−91 ± 4−310 ± 222.5 ± 0.11.4 ± 0.3201NDNDNDNDErrors are given as the standard error of the fitted parameter except for the standard errors of run length of GFP-Ncd that were calculated from the raw data. ND, not determined. Open table in a new tab Figure 4Model of Processive Movement of NcdShow full caption(A) Schematic drawings of Ncd moving on a single microtubule (not to scale). In the strong binding state, Ncd binds rigidly to the MT, but in the weak binding state, it can move diffusively without detaching from the MT. The directional bias could be made by a “power stroke” or biased affinity toward the minus end of the MT while being tethered to it. The E-hooks are drawn for only one protofilament for simplicity.(B) Processive movement of Ncd enhanced by MT bundling.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Errors are given as the standard error of the fitted parameter except for the standard errors of run length of GFP-Ncd that were calculated from the raw data. ND, not determined. (A) Schematic drawings of Ncd moving on a single microtubule (not to scale). In the strong binding state, Ncd binds rigidly to the MT, but in the weak binding state, it can move diffusively without detaching from the MT. The directional bias could be made by a “power stroke” or biased affinity toward the minus end of the MT while being tethered to it. The E-hooks are drawn for only one protofilament for simplicity. (B) Processive movement of Ncd enhanced by MT bundling. In this report, we provide the first evidence of a processive movement exhibited by individual minus-end-directed kinesins by using a single-molecule imaging technique. Our results reveal that Ncd exhibited a distinct processivity from dimeric kinesin-1, containing a diffusive element that does not require ATP hydrolysis (Figure 4A). Such weak processivity also has been reported for KIF1A [18Okada Y. Hirokawa N. A processive single-headed motor: Kinesin superfamily protein KIF1A.Science. 1999; 283: 1152-1157Crossref PubMed Scopus (335) Google Scholar], Eg5 [20Kwok B.H. Kapitein L.C. Kim J.H. Peterman E.J. Schmidt C.F. Kapoor T.M. Allosteric inhibition of kinesin-5 modulates its processive directional motility.Nat. Chem. Biol. 2006; 2: 480-485Crossref PubMed Scopus (88) Google Scholar], and monomeric kinesin-1 [17Inoue Y. Iwane A.H. Miyai T. Muto E. Yanagida T. Motility of single one-headed kinesin molecules along microtubules.Biophys. J. 2001; 81: 2838-2850Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar]. The Ncd tail serves as a tether to MTs and thereby enables Ncd to behave as a processive motor, although the coupling between the steps and the ATP turnover would be looser compared with dimeric kinesin-1. However, even in dimeric kinesin-1, electrostatic interaction is reported to be important for processivity [21Thorn K.S. Ubersax J.A. Vale R.D. Engineering the processive run length of the kinesin motor.J. Cell Biol. 2000; 151: 1093-1100Crossref PubMed Scopus (226) Google Scholar]. The electrostatic tethering mechanism might therefore be a general principle for processivity in both vesicle transporters and spindle motors. Why, then, is Ncd not highly processive like the vesicle transporter kinesin-1? We found that individual GFP-Ncd molecules can modulate its processivity depending on MT bundling (Figure 4B). A recent study revealed that the turnover rate of Ncd on MT bundles is considerably high in vivo [10Goshima G. Nedelec F. Vale R.D. Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.J. Cell Biol. 2005; 171: 229-240Crossref PubMed Scopus (195) Google Scholar], suggesting that Ncd is a dynamic, not a static, crosslinker. Our results may reinforce this emerging view and provide further understanding of spindle formation. We speculate that the variable processivity of Ncd may reflect the physiological needs to dynamically assemble the spindle structure. The diffusive nature of Ncd in the weak binding state may allow Ncd to wait on single MTs without consuming ATP. When the MTs begin to be organized, Ncd would move with enhanced processivity along bundled MTs toward the minus end, forming transient crosslinks and sliding MTs. In this model, Ncd and the MTs form a feedback loop, namely by which more Ncd molecules would be activated by increasing numbers of bundled MTs, because the MT bundle is a product of crosslinking by Ncd itself. However, it remains unclear how Ncd molecules exert the force required to slide MTs. Previous studies have shown that self-organization of MT asters requires processive motors [28Nedelec F.J. Surrey T. Maggs A.C. Leibler S. Self-organization of microtubules and motors.Nature. 1997; 389: 305-308Crossref PubMed Scopus (578) Google Scholar, 29Surrey T. Nedelec F. Leibler S. Karsenti E. Physical properties determining self-organization of motors and microtubules.Science. 2001; 292: 1167-1171Crossref PubMed Scopus (411) Google Scholar], but individual dimeric Ncd molecules cannot step processively against the load [30deCastro M.J. Fondecave R.M. Clarke L.A. Schmidt C.F. Stewart R.J. Working strokes by single molecules of the kinesin-related microtubule motor ncd.Nat. Cell Biol. 2000; 2: 724-729Crossref PubMed Scopus (67) Google Scholar]. The discrepancy could be resolved if small numbers of Ncd molecules acted cooperatively to generate the required force, as previously reported [16deCastro M.J. Ho C.H. Stewart R.J. Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive.Biochemistry. 1999; 38: 5076-5081Crossref PubMed Scopus (59) Google Scholar, 29Surrey T. Nedelec F. Leibler S. Karsenti E. Physical properties determining self-organization of motors and microtubules.Science. 2001; 292: 1167-1171Crossref PubMed Scopus (411) Google Scholar, 31Badoual M. Julicher F. Prost J. Bidirectional cooperative motion of molecular motors.Proc. Natl. Acad. Sci. USA. 2002; 99: 6696-6701Crossref PubMed Scopus (156) Google Scholar, 32Sciambi C.J. Komma D.J. Skold H.N. Hirose K. Endow S.A. A bidirectional kinesin motor in live Drosophila embryos.Traffic. 2005; 6: 1036-1046Crossref PubMed Scopus (9) Google Scholar]. The processivity of individual Ncd molecules at the low loads that we have shown here may be advantageous for efficient accumulation of Ncd and may lead to cooperative force generation in the ensemble by increasing local Ncd concentration. The variable processivity may be tuned such that crosslinks neither become too static nor too dynamic to efficiently assemble/disassemble the spindle. Further study on the cooperative behavior of spindle motors is needed to elucidate the underlying mechanisms. This work was supported by a grant-in-aid for scientific research (B) from the Japan Society for the Promotion of Science (JSPS); a grant-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT); a Core Research for Evolutional Science and Technology (CREST) program grant from the Japan Science and Technology Agency (JST); and a grant-in-aid for JSPS fellows from JSPS. Download .pdf (.84 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Seven Figures, and Two Tables Download .mov (2.2 MB) Help with mov files Movie S1. Single-Molecule Motility of GFP-Ncd26 along a Single MT (20× Real Time)The movement was observed in assay buffer containing 1 mM ATP and 5 mM KAc. GFP-Ncd26 moved processively along the single MT in a diffusive manner. The frames from a continuous GFP recording (green) were overlaid on one Cy5 image of the MT (red). Download .mov (3.35 MB) Help with mov files Movie S2. Single-Molecule Motility of GFP-Ncd195 along Parallel MT Bundles (10× Real Time)The movement was observed in assay buffer containing 1 mM ATP and 105 mM KAc. The frames from a continuous GFP recording (green) were overlaid on one Cy5 image of the MTs (red) and one BODIPY FL image of the axonemes. Download .mov (2.49 MB) Help with mov files Movie S3. Single-Molecule Motility of GFP-Ncd195 along an Axoneme (10× Real Time)The movement was observed in assay buffer containing 1 mM ATP and 105 mM KAc. The frames from a continuous GFP recording (green) were overlaid on one Cy5 image of the axoneme (red)." @default.
• W2025753616 created "2016-06-24" @default.
• W2025753616 creator A5006791406 @default.
• W2025753616 creator A5065125586 @default.
• W2025753616 date "2008-01-01" @default.
• W2025753616 modified "2023-10-14" @default.
• W2025753616 title "Minus-End-Directed Motor Ncd Exhibits Processive Movement that Is Enhanced by Microtubule Bundling In Vitro" @default.
• W2025753616 cites W1503265874 @default.
• W2025753616 cites W1569667199 @default.
• W2025753616 cites W1585309689 @default.
• W2025753616 cites W1968837995 @default.
• W2025753616 cites W1970897399 @default.
• W2025753616 cites W1975928024 @default.
• W2025753616 cites W1976445049 @default.
• W2025753616 cites W1989829257 @default.
• W2025753616 cites W1996363040 @default.
• W2025753616 cites W1999990117 @default.
• W2025753616 cites W2000364651 @default.
• W2025753616 cites W2005261382 @default.
• W2025753616 cites W2015140949 @default.
• W2025753616 cites W2016518842 @default.
• W2025753616 cites W2029189987 @default.
• W2025753616 cites W2035354410 @default.
• W2025753616 cites W2046320664 @default.
• W2025753616 cites W2062496406 @default.
• W2025753616 cites W2072495256 @default.
• W2025753616 cites W2086227169 @default.
• W2025753616 cites W2086544026 @default.
• W2025753616 cites W2101613161 @default.
• W2025753616 cites W2102340487 @default.
• W2025753616 cites W2106258850 @default.
• W2025753616 cites W2113170636 @default.
• W2025753616 cites W2125394005 @default.
• W2025753616 cites W2127645433 @default.
• W2025753616 cites W2130217713 @default.
• W2025753616 cites W2149578350 @default.
• W2025753616 cites W2154782261 @default.
• W2025753616 cites W2162632054 @default.
• W2025753616 cites W2168166944 @default.
• W2025753616 doi "https://doi.org/10.1016/j.cub.2007.12.056" @default.
• W2025753616 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18207739" @default.
• W2025753616 hasPublicationYear "2008" @default.
• W2025753616 type Work @default.
• W2025753616 sameAs 2025753616 @default.
• W2025753616 citedByCount "81" @default.
• W2025753616 countsByYear W20257536162012 @default.
• W2025753616 countsByYear W20257536162013 @default.
• W2025753616 countsByYear W20257536162014 @default.
• W2025753616 countsByYear W20257536162015 @default.
• W2025753616 countsByYear W20257536162016 @default.
• W2025753616 countsByYear W20257536162017 @default.
• W2025753616 countsByYear W20257536162018 @default.
• W2025753616 countsByYear W20257536162019 @default.
• W2025753616 countsByYear W20257536162020 @default.
• W2025753616 countsByYear W20257536162021 @default.
• W2025753616 countsByYear W20257536162022 @default.
• W2025753616 crossrefType "journal-article" @default.
• W2025753616 hasAuthorship W2025753616A5006791406 @default.
• W2025753616 hasAuthorship W2025753616A5065125586 @default.
• W2025753616 hasBestOaLocation W20257536161 @default.
• W2025753616 hasConcept C121332964 @default.
• W2025753616 hasConcept C12554922 @default.
• W2025753616 hasConcept C160408235 @default.
• W2025753616 hasConcept C169760540 @default.
• W2025753616 hasConcept C202751555 @default.
• W2025753616 hasConcept C20418707 @default.
• W2025753616 hasConcept C24890656 @default.
• W2025753616 hasConcept C2780226923 @default.
• W2025753616 hasConcept C54355233 @default.
• W2025753616 hasConcept C59006786 @default.
• W2025753616 hasConcept C86803240 @default.
• W2025753616 hasConcept C93126451 @default.
• W2025753616 hasConcept C95444343 @default.
• W2025753616 hasConceptScore W2025753616C121332964 @default.
• W2025753616 hasConceptScore W2025753616C12554922 @default.
• W2025753616 hasConceptScore W2025753616C160408235 @default.
• W2025753616 hasConceptScore W2025753616C169760540 @default.
• W2025753616 hasConceptScore W2025753616C202751555 @default.
• W2025753616 hasConceptScore W2025753616C20418707 @default.
• W2025753616 hasConceptScore W2025753616C24890656 @default.
• W2025753616 hasConceptScore W2025753616C2780226923 @default.
• W2025753616 hasConceptScore W2025753616C54355233 @default.
• W2025753616 hasConceptScore W2025753616C59006786 @default.
• W2025753616 hasConceptScore W2025753616C86803240 @default.
• W2025753616 hasConceptScore W2025753616C93126451 @default.
• W2025753616 hasConceptScore W2025753616C95444343 @default.
• W2025753616 hasFunder F4320320912 @default.
• W2025753616 hasFunder F4320334764 @default.
• W2025753616 hasFunder F4320334789 @default.
• W2025753616 hasFunder F4320338075 @default.
• W2025753616 hasIssue "2" @default.
• W2025753616 hasLocation W20257536161 @default.
• W2025753616 hasLocation W20257536162 @default.
• W2025753616 hasOpenAccess W2025753616 @default.
• W2025753616 hasPrimaryLocation W20257536161 @default.
• W2025753616 hasRelatedWork W1964816796 @default.
• W2025753616 hasRelatedWork W1974528900 @default.
• W2025753616 hasRelatedWork W1994811243 @default.

• next