Microtubule-associated motility in cytoplasmic extracts of sea urchin eggs.
We have developed a method for producing sea urchin egg cytoplasmic extracts which support substantial microtubule-associated motility, particularly minus end-directed motility characteristic of cytoplasmic dynein. Particles translocated along microtubules and axonemes predominantly in the minus end direction; microtubules and axonemes glided across the coverslip surface only in the plus end direction (as expected for a minus-end directed motor bound to the coverslip surface); and microtubules crosslinked into bundles in an antiparallel orientation. Velocities of particle and microtubule translocation were in the range of 0.5-1.8 microns/sec. Vanadate at 10 microM inhibited all gliding of the microtubules and axonemes, yet bidirectional particle transport persisted. Vanadate at concentrations of 25 microM and higher inhibited nearly all microtubule-based motility in the preparation and produced parallel bundling of the microtubules. Motility was slowed but not stopped in the presence of 5 mM AMP-PNP. Usually when a particle bound to a microtubule wall, it moved to the microtubule minus end. These particles often remained attached to the minus end. When a microtubule plus end in the shortening phase of dynamic instability reached a stationary particle on the microtubule, sometimes normal minus end-directed motility was activated, or at other times the particle remained attached to the shortening plus end.
Gliksman
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NR
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Salmon
E D
ED
D000818:Animals; D002021:Buffers; D002457:Cell Extracts / Q000502:physiology*; D003593:Cytoplasm / Q000502:physiology*; D004398:Dyneins / Q000502:physiology*; D005260:Female; D006531:HEPES; D007399:Interphase / Q000502:physiology; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D010063:Ovum; D012617:Sea Urchins
D002021:Buffers; D002457:Cell Extracts; D004398:Dyneins; D006531:HEPES