RESUMEN
Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (â¼ 4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein before ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins.
Asunto(s)
Dineínas Axonemales/metabolismo , Dineínas Citoplasmáticas/metabolismo , Dineínas/metabolismo , Erizos de Mar/metabolismo , Animales , Microtúbulos/metabolismo , MovimientoRESUMEN
Dynein transduces the chemical energy of ATP hydrolysis into mechanical work through conformational changes. To identify the factors governing the coupling between the ATPase activity and the motile activity of the dynein molecule, we examined the effects of potassium iodide, which can unfold protein tertiary structures, on dynein activity in reactivated sea urchin sperm flagella. The presence of low concentrations of KI (0.05-0.1 M) in the reactivating solution did not influence the stable beating of demembranated flagella at 0.02-1 mM ATP, when the total concentration of potassium was kept at 0.15 M by adding K-acetate. However, double-reciprocal plots of ATP concentration and beat frequency showed a mixed type of inhibition by KI, indicating the possibility that KI inhibits the ATP hydrolysis and decreases the maximum sliding velocity. The ATPase activity of 21S dynein with or without microtubules did not decrease with the KI concentration. In the elastase-treated axonemes, KI decreased the velocity of sliding disintegration, while it increased the frequency of occurrence of axonemes showing no sliding. This may be related to some defect in the coordination of dynein activities. On 21S dynein adsorbed on a glass surface, however, the velocity of microtubule sliding was increased by KI, while KI lowered the dynein-microtubule affinity. The velocity further increased under lower salt conditions enhancing the dynein-microtubule interactions. The results suggest the importance of organized regulation of the dynamic states of dynein-microtubule interactions through the stalk for the coupling between the ATPase activity and the motile activity of dynein in beating flagella.
Asunto(s)
Adenosina Trifosfato/metabolismo , Dineínas Axonemales/metabolismo , Yoduros/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Masculino , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Erizos de Mar/efectos de los fármacos , Erizos de Mar/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismoRESUMEN
ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.0-7.2 which is lower than the physiological pH of 8, about 90% of reactivated flagella were motionless at 1 mM ATP, while about 60% were motile at 0.02 mM ATP. The motionless flagella at 1 mM ATP maintained a single large bend or an S-shaped bend, indicating formation of dynein crossbridges in the axoneme. The ATP-dependent inhibition of flagellar movement was released by ADP, and was absent in outer arm-depleted flagella. Similar inhibition was also observed at 0.02 mM ATP when demembranated flagella were reactivated in the presence of Li+ or pretreated with protein phosphatase 1 (PP1). ADP also released this type of ATP-inhibition. In PP1-pretreated axonemes the binding of a fluorescent analogue of ADP to dynein decreased. Under elastase-treatment at pH 8.0, the beating of demembranated flagella at 1 mM ATP and 0.02 mM ATP lasted for approximately 100 and 45 s, respectively. The duration of beating at 0.02 mM ATP was prolonged by Li+, and that at 1 mM ATP was shortened by removal of outer arms. These results indicate that the regulation of on/off switching of dynein motile activity of flagella involves ATP-induced inhibition and ADP-induced activation, probably through phosphorylation/dephosphorylation of outer arm-linked protein(s).
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Erizos de Mar/fisiología , Cola del Espermatozoide/fisiología , Adenosina Trifosfato/efectos adversos , Adenosina Trifosfato/análogos & derivados , Animales , Dineínas/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Microtúbulos/metabolismo , Proteína Fosfatasa 1/metabolismo , Motilidad Espermática/efectos de los fármacos , Cola del Espermatozoide/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/fisiologíaRESUMEN
The movement of eukaryotic flagella and cilia is regulated by intracellular calcium. We have tested a model in which the central pair of microtubules mediate the effect of Ca(2+) to modify the dynein activity. We used a novel microtubule sliding assay that allowed us to test the effect of Ca(2+) in the presence or absence of the central-pair microtubules. When flagellar axonemes of sea-urchin sperm were exposed to ATP in the presence of elastase, they showed different types of sliding disintegration depending on the ATP concentration: at low concentrations of ATP (=50 micro M), all the axonemes were disintegrated into individual doublets by microtubule sliding; by contrast, at high ATP concentrations (>/=100 micro M), a large proportion of the axonemes showed limited sliding and split lengthwise into a pair of two microtubule bundles, one of which was thicker than the other. The sliding behaviour of the axonemes was also influenced by Ca(2+). Thus, at 1 mM ATP, the proportion of axonemes that split into two bundles increased from 25% at <10(-9) M Ca(2+) to 60% at 10(-4) M Ca(2+), whereas the sliding velocity of doublets during the splitting did not change. Electron microscopy of split bundles showed that the thicker bundles contained five or six doublets and the central pair, whereas the thinner bundles contained three or four doublets but not the central pair. Closer examinations revealed that the thicker bundles were dominated by four patterns of doublet combinations: doublets 8-9-1-2-3-4, 8-9-1-2-3, 4-5-6-7-8 and 3-4-5-6-7-8. This indicates that the sliding occurred preferentially at one or two fixed interdoublet sites on either side of the central-pair microtubules, whereas the sliding at the remaining interdoublet sites was inhibited under these conditions. Ca(2+) reduced the appearance of the 4-5-6-7-8 and 3-4-5-6-7-8 patterns and increased the 8-9-1-2-3-4 and 8-9-1-2-3 patterns. The splitting patterns are possibly related to the switching mechanism of the dynein activity underlying the cyclical flagellar bending. To investigate the role of the central pair in the regulation of the dynein activity by Ca(2+), we studied the behaviour of singlet microtubules applied to the dynein arms exposed on the doublets of the split bundles that were either associated with the central pair or not. Microtubules moved along both the thicker and the thinner bundles but the frequency of microtubule sliding on the thinner (i.e. the central-pair-less) bundles was three to four times (at =10(-5) M Ca(2+)) and ten times (at 10(-4) M Ca(2+)) as large as that on the thicker, central-pair-associated bundles. Furthermore, the velocity of microtubule sliding at 1 mM ATP on the thicker bundles were significantly reduced by 10(-7)-10(-4) M Ca(2+), whereas that on the thinner bundles was not changed by the concentration of Ca(2+). These results indicate that Ca(2+) inhibits the activity of dynein arms on the doublets through a regulatory mechanism that involves the central pair and the radial spoke complex. This mechanism might control the switching of the dynein activity within the axoneme to induce the oscillatory bending movement of the flagellum.