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Biophysical Journal May 2014We 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...
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.
Topics: Adenosine Triphosphatases; Axonemal Dyneins; Chlamydomonas; Flagella; Kinetics; Microtubules; Movement; Mutation; Plant Proteins; Protein Transport
PubMed: 24853744
DOI: 10.1016/j.bpj.2014.04.009 -
Nature Structural & Molecular Biology Jul 2017
Topics: Dyneins; Models, Biological; Protein Conformation; Protein Multimerization; Protein Transport
PubMed: 28686228
DOI: 10.1038/nsmb.3429 -
Biochimie Mar 2000Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules....
Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.
Topics: Adenosine Triphosphate; Animals; Blotting, Western; Cattle; Chromatography, Gel; Cytoplasm; Dynactin Complex; Dyneins; Electrophoresis, Polyacrylamide Gel; Microtubule-Associated Proteins; Microtubules; Neurospora crassa
PubMed: 10863006
DOI: 10.1016/s0300-9084(00)00206-6 -
Andrologia May 2016Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the...
Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+).
Topics: Axonemal Dyneins; Cadmium; Calcium-Transporting ATPases; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Assays; Healthy Volunteers; Humans; Male; Sperm Motility; Spermatozoa
PubMed: 26259968
DOI: 10.1111/and.12466 -
Cell Motility and the Cytoskeleton Jan 2001The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction...
The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.
Topics: Adenosine Triphosphate; Animals; Blotting, Western; Brain Chemistry; Dynactin Complex; Dyneins; Enzyme Activation; Isoenzymes; Male; Microtubule-Associated Proteins; Potassium Iodide; Protein Subunits; Rats; Rats, Sprague-Dawley; Testis
PubMed: 11124710
DOI: 10.1002/1097-0169(200101)48:1<52::AID-CM5>3.0.CO;2-X -
Journal of Supramolecular Structure 1979The effects of thiourea and of several substituted thioureas -- phenylthiourea, alpha-naphtylthiourea, metiamide, and burimamide -- on dynein ATPase have been studied....
The effects of thiourea and of several substituted thioureas -- phenylthiourea, alpha-naphtylthiourea, metiamide, and burimamide -- on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of beta-mercaptoethanol or dithiothreitol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (approximately 1 microM) concentrations of ATP and, less effectively, by AMP-PNP, but not by AMP-PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP-PNP. The thioureas inhibit the ATP-induced decrease in turbidity (measured as delta A350) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of beta-mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice-extracted axonemes is not changed by treatment with phenylthiourea or metiamide. These observations indicate that the thioureas react with at least two sets of SH or S--S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.
Topics: Adenosine Triphosphatases; Animals; Burimamide; Cilia; Dyneins; Kinetics; Metiamide; Naphthalenes; Nephelometry and Turbidimetry; Phenylthiourea; Structure-Activity Relationship; Tetrahymena pyriformis; Thiourea
PubMed: 161792
DOI: 10.1002/jss.400120104 -
Biophysical Journal Apr 2020Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor...
Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein exhibits various bipedal stepping motions: inchworm and hand-over-hand motions, as well as nonalternating steps of one head. However, the molecular basis to achieve such diverse stepping manners remains unclear because of the lack of an experimental method to observe stepping and the ATPase reaction of dynein simultaneously. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and perform Gillespie Monte Carlo simulations that qualitatively reproduce most experimental data obtained to date. The model represents the status of each motor domain as five states according to conformation and nucleotide- and microtubule-binding conditions of the domain. In addition, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, similarly to experimental data. The model reproduced key experimental motility-related properties, including velocity and run length, as functions of the ATP concentration and external force, therefore providing a plausible explanation of how dynein achieves various stepping manners with explicit characterization of nucleotide states. Our model highlights the uniqueness of dynein in the coupling of ATPase with its movement during both inchworm and hand-over-hand stepping.
Topics: Adenosine Triphosphate; Cytoplasmic Dyneins; Dyneins; Hydrolysis; Kinetics; Microtubules
PubMed: 32272056
DOI: 10.1016/j.bpj.2020.03.012 -
The Journal of Cell Biology Sep 1988We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro...
We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.
Topics: Adenosine Triphosphatases; Animals; Brain; Centrifugation, Density Gradient; Cytosol; Dyneins; Microtubule-Associated Proteins; Microtubules; Oxidation-Reduction; Potassium Chloride; Substrate Specificity; Tubulin
PubMed: 2971069
DOI: 10.1083/jcb.107.3.1001 -
Cell Motility and the Cytoskeleton Jul 2002The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these...
The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.
Topics: Amino Acid Sequence; Animals; Chlamydomonas; Dithionitrobenzoic Acid; Dyneins; Flagella; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Protein Structure, Tertiary; Sequence Alignment; Structure-Activity Relationship
PubMed: 12112141
DOI: 10.1002/cm.10044 -
Biochemical and Biophysical Research... Jan 1982
Topics: Actomyosin; Adenine; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Dyneins; Immunosuppressive Agents; Kinetics; Male; Myosins; Sea Urchins; Spermatozoa
PubMed: 6462140
DOI: 10.1016/0006-291x(82)91964-7