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1.
We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s–1 and kcat,MT = 1.09 s–1, respectively) were lower than those of dynein c (kcat = 1.75 s–1 and kcat,MT = 2.03 s–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke. 相似文献
2.
Youské Shimizu Hitoshi Sakakibara Hiroaki Kojima Kazuhiro Oiwa 《Biophysical journal》2014,106(10):2157-2165
We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s–1 and kcat,MT = 1.09 s–1, respectively) were lower than those of dynein c (kcat = 1.75 s–1 and kcat,MT = 2.03 s–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke. 相似文献
3.
Kenji Kikushima 《Biophysical journal》2009,97(6):1657-1662
To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as ∼0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme. 相似文献
4.
Axonemal dyneins provide the driving force for flagellar/ciliary bending. Nucleotide-induced conformational changes of flagellar dynein have been found both in vitro and in situ by electron microscopy, and in situ studies demonstrated the coexistence of at least two conformations in axonemes in the presence of nucleotides (the apo and the nucleotide-bound forms). The distribution of the two forms suggested cooperativity between adjacent dyneins on axonemal microtubule doublets. Although the mechanism of such cooperativity is unknown it might be related to the mechanism of bending. To explore the mechanism by which structural heterogeneity of axonemal dyneins is induced by nucleotides, we used cilia from Tetrahymena thermophila to examine the structure of dyneins in a) the intact axoneme and b) microtubule doublets separated from the axoneme, both with and without additional pure microtubules. We also employed an ATPase assay on these specimens to investigate dynein activity functionally. Dyneins on separated doublets show more activation by nucleotides than those in the intact axoneme, both structurally and in the ATPase assay, and this is especially pronounced when the doublets are coupled with added microtubules, as expected. Paralleling the reduced ATPase activity in the intact axonemes, a lower proportion of these dyneins are in the nucleotide-bound form. This indicates a coordinated suppression of dynein activity in the axoneme, which could be the key for understanding the bending mechanism. 相似文献
5.
We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules. 相似文献
6.
The translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. Dynein was first discovered in the ciliary axoneme, where it causes the directed sliding between outer doublet microtubules that underlies ciliary bending. The initiation and propagation of ciliary bends are produced by a precisely located array of different dyneins containing eight or more different dynein heavy chain isoforms. The detailed clarification of the structural and functional diversity of axonemal dynein heavy chains will not only provide the key to understanding how cilia function, but also give insights applicable to the study of non-axonemal microtubule motors. 相似文献
7.
Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins. 相似文献
8.
We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 microns in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 microns/s. The addition of 0.1% Nonidet P-40 was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10(-9) M Ca, and at 0.05 to 1 mM ATP in the presence of 10(-3) M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2-8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility. 相似文献
9.
Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of eukaryotic cilia and flagella. Recent structural studies of the axoneme, which forms the core of cilia and flagella, have used cryo-electron tomography to reveal new details of the interactions between some of the multitude of proteins that form the axoneme and regulate its movement. Connections between the several types of dyneins, in particular, suggest ways in which their action might be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding mechanical properties related to the bending of the axoneme, and has also offered insight into the potential role of doublets in the mechanism of dynein activity regulation. 相似文献
10.
We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 μm in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 μm/s. The addition of 0.1% Nonidet P-4O was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10−9 M Ca, and at 0.05 to 1 mM ATP in the presence of 10−3 M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2–8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility. 相似文献
11.
Triscia W. Hendrickson Jonathan L. Goss Charles A. Seaton Henry W. Rohrs 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):3265-3271
Dyneins are minus end directed microtubule motors that play a critical role in ciliary and flagellar movement. Ciliary dyneins, also known as axonemal dyneins, are characterized based on their location on the axoneme, either as outer dynein arms or inner dynein arms. The I1 dynein is the best-characterized subspecies of the inner dynein arms; however the interactions between many of the components of the I1 complex and the axoneme are not well defined. In an effort to elucidate the interactions in which the I1 components are involved, we performed zero-length crosslinking on axonemes and studied the crosslinked products formed by the I1 intermediate chains, IC138 and IC140. Our data indicate that IC138 and IC140 bind directly to microtubules. Mass-spectrometry analysis of the crosslinked product identified both α- and β-tubulin as the IC138 and IC140 binding partners. This was further confirmed by crosslinking experiments carried out on purified I1 fractions bound to Taxol-stabilized microtubules. Furthermore, the interaction between IC140 and tubulin is lost when IC138 is absent. Our studies support previous findings that intermediate chains play critical roles in the assembly, axonemal targeting and regulation of the I1 dynein complex. 相似文献
12.
Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein. 相似文献
13.
The movement of eukaryotic flagella is characterized by its oscillatory nature. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar axoneme. Although the mechanism regulating the dynein activity is unknown, previous studies have suggested that the flagellar bending itself is important in the feedback mechanism responsible for the oscillatory bending. If so, experimentally bending the microtubules would be expected to affect the sliding activity of dynein. Here we report on experiments with bundles of doublets obtained by inducing sliding in elastase-treated axonemes. Our results show that bending not only "switches" the dynein activity on and off but also affects the microtubule sliding velocity, thus supporting the idea that bending is involved in the self-regulatory mechanism underlying flagellar oscillation. 相似文献
14.
Recently Vale et al. (1989, Cell 59, 915-925.) reported an observation of the one-dimensional Brownian movement of microtubules bound to flagellar dynein through a weak-binding interaction. In this study, we propose a theoretical model of this phenomenon. Our model consists of a rigid microtubule associated with a number of elastic dynein heads through a weak-binding interaction at equilibrium. The model implies that (1) the Brownian motion of the microtubule is not directly driven by the atomic collision of the solvent particles, but is driven by the thermally-generated structural fluctuations of the dynein heads which interact with the microtubule; (2) dynein heads through a weak-binding interaction exert a frictional drag force on the sliding motion of the microtubule and the drag force is proportional to the sliding velocity the same as in hydrodynamic viscous friction. This protein friction, with such viscous-like characteristics, may well play a role as a velocity-limiting factor in the normal ATP-induced sliding movement of motile proteins. 相似文献
15.
To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein. 相似文献
16.
Structural-functional relationships of the dynein,spokes, and central-pair projections predicted from an analysis of the forces acting within a flagellum
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Lindemann CB 《Biophysical journal》2003,84(6):4115-4126
In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25-1.0 nN/ micro m, with 0.5 nN/ micro m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also approximately 0.5 nN. The equivalence of the maximum dynein force/ micro m and t-force/ micro m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure. 相似文献
17.
Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement. 相似文献
18.
Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N + 1) tipward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. The 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg2+, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein. 相似文献
19.
JØRGEN LARSEN KURT BARKALOW TOSHIKAZU HAMASAKI PETER SATIR 《The Journal of eukaryotic microbiology》1991,38(1):55-61
ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg2- , ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein. 相似文献
20.
Brokaw CJ 《Cell motility and the cytoskeleton》1999,42(2):134-148
Outer arm dynein removal from flagella by genetic or chemical methods causes decreased frequency and power, but little change in bending pattern. These results suggest that outer arm dynein operates within bends to increase the speed of bend propagation, but does not produce forces that alter the bending pattern established by inner arm dyneins. A flagellar model incorporating different cross-bridge models for inner and outer arm dyneins has been examined. The inner arm dynein model has a hyperbolic force-velocity curve, with a maximum average force at 0 sliding velocity of about 14 pN for each 96 nm group of inner arm dyneins. The outer arm dynein model has a very different force-velocity curve, with a maximum force at about 10-15% of V(max). The outer arm dynein model is adjusted so that the unloaded sliding velocity for a realistic mixture of inner and outer arm dyneins is twice the unloaded sliding velocity for the inner arm dynein model alone. With these cross-bridge models, a flagellar model can be obtained that reduces its sliding velocity and frequency by approximately 50% when outer arm dyneins are removed, with little change in bending pattern. The addition of outer arm dyneins, therefore, gives an approximately 4-fold increase in power output against viscous resistances, and outer arm dyneins may generate 90% or more of the power output. Cell Motil. 相似文献