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|Title:||Microtubule-based vesicle transport and isolation of a microtubule-based motor from Paramecium|
|Authors:||Schroeder, Christopher C.|
|Keywords:||Paramecium -- Microbiology|
|Abstract:||Cytoplasmic microtubule-based motility in Paramecium was investigated using a variety of techniques. Vesicle transport along microtubules was directly observed using video enhanced contrast microscopy. The observation of the association of vesicles with microtubules at the ultrastructural level was accomplished with the quick-freeze, deep-etch technique. Finally, a microtubule-based motor from the cytosol was isolated and purified. Two microtubular structures, the cytopharyngeal microtubular ribbons and the post oral fibers, supported vesicle transport, but the properties of transport along these two microtubular structures were quite different. Each cytopharyngeal microtubular ribbon is composed of 10 - 12 microtubules aligned in a single plane oriented perpendicularly to the oral apparatus. The post oral fibers are composed of several bundles of microtubules which originate at the right side of the oral apparatus and extend to the posterior end of the cell. Along the microtubular ribbons vesicles moved primarily toward the oral apparatus, a minus-end directed movement, at a rate of approximately 6 μm/sec. Only three populations of vesicles (discoidal vesicles, acidosomes, and 100 nm vesicles) were transported along these microtubules. This transport was smooth, continuous and exclusively on the anterior side of the ribbons. Vesicles occasionally moved away from the oral apparatus, but in this direction they moved only along the posterior side of the microtubular ribbons. The smooth nature and sidedness of transport are unique findings for an in vivo system. Supporting evidence for the above observations was found at the ultrastructural level. Discoidal vesicles, acidosomes, and 100 nm vesicles were aligned in single file along the anterior side of the microtubular ribbons. Cross bridges were evident in most cases between the vesicle membrane and the microtubules. Transport along the post oral fibers occurred only during and shortly after digestive vacuole formation and release from the oral apparatus. A distinct on/off switching mechanism was evident. The rate of transport was approximately 20 urn/sec and exclusively plus-end directed. In contrast to transport along the microtubular ribbons, a heterogeneous population of vesicles were transported along the post oral fibers. To understand the molecular basis for microtubule-based vesicle transport, a microtubule-based motor was isolated from the cytosol using bovine brain micro tubules as an affinity matrix. This motor was found to be a two headed cytoplasmic dynein which closely resembled the cytoplasmic dyneins of higher organisms. The dynein exhibited a microtubule-stimulated ATPase activity and promoted microtubule gliding in an in vitro assay. This cytoplasmic dynein was directly compared to axonemal dyneins from Paramecium and were found to differ by five separate criteria. The criteria were morphology, sedimentation coefficient, polypeptide composition, vanadate cleavage patterns and CTPase/ATPase ratio. Therefore, the cytoplasmic dynein was not an axonemal dynein precursor. This dynein may be the motor that drives the microtubule-based vesicle transport along the ribbons in minus-end directed movements.|
Thesis (Ph. D.)--University of Hawaii at Manoa, 1990.
Includes bibliographical references (leaves 120-132)
xi, 132 leaves, bound ill. 29 cm
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|Appears in Collections:||Ph.D. - Microbiology|
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