Stabilization of microtubules by dynein-binding in vitro. Stability of microtubule-dynein complex.
We have studied the effects of dynein binding on the stability of microtubules in vitro, using Tetrahymena ciliary dynein and microtubules (three-cycled purified microtubules: 3 X-Mts and phosphocellulose-column purified microtubules: PC-Mts). To determine the relative stability of the microtubules, we first prepared the microtubules bound with dynein (Mts--dynein complex) and subjected the Mts-dynein complex to treatments that depolymerize the microtubules, such as dilution to below critical concentration of tubulin, calcium ions and lower temperature. Dark-field microscopy revealed that the microtubules in the Mts--dynein complex appeared intact under conditions which otherwise result in microtubule depolymerization. However, when dynein was dissociated from the Mts--dynein complex with addition of ATP, no microtubule was found in the specimens under the same conditions. That is, the microtubules in the Mts--dynein complex did not depolymerize upon dilution with the buffer solution to below critical concentration of tubulin. However, addition of ATP to the diluted specimen caused dynein to become separated from the Mts, resulting in complete depolymerization of the microtubules. Stability of the microtubules was also studied by the turbidity changes and was confirmed by the patterns of stained gel bands in electrophoresis. With the addition of calcium ion, the Mts--dynein complex decomposed into separate molecules dynein and tubulin. At the lower temperature of 0 degrees C, the 3 X-Mts--dynein complex was decomposed into dynein and tubulin, while the microtubules in the PC-Mts--dynein complex did not depolymerize. Although we have not yet studied the effects of cytoplasmic dynein binding on the microtubules, the results suggest that the stabilizing effect of dynein binding to the microtubules is one of the important functions of dynein in vivo.
Ohba
S
S
Department of Biology, Ochanomizu University, Tokyo, Japan.
Kamata
K
K
Miki-Noumura
T
T
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004398:Dyneins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D013696:Temperature; D016808:Tetrahymena thermophila / Q000737:chemistry; D014404:Tubulin / Q000302:isolation & purification
D014404:Tubulin; D004398:Dyneins; D002118:Calcium