Dynein decoration of microtubules--determination of polarity.
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Adenosine Triphosphatases↗
Biomedical subjects
L T Haimo
Publications and source records attributed to L T Haimo.
Dynein binding to microtubules containing microtubule-associated proteins.
Microtubule-associated proteins (MAPs), isolated from brain tubulin, bound to and saturated outer fibers of Chlamydomonas flagella. MAPs present on these microtubules prevented the subsequent recombination of dynein. MAPs also bound to intact axonemes and thus did not specifically bind to the dynein binding sites on the A subfiber. A molar ratio of 1 mole MAP2 per 27 moles tubulin dimers at saturation of the outer fibers with MAP2 suggested that MAPs could effectively interfere with dynein recombination only if the MAPs were near the dynein binding sites to sterically prevent binding. However, electron microscopic observations indicated that MAPs were not localized but, instead, were dispersed around the outer fibers. In addition, MAP2 present at saturating amounts on in vitro assembled brain microtubules had no significant effect on dynein binding. Dynein-decorated microtubules contained clusters of arms suggesting that there may be cooperative interaction between the arms during dynein binding. Because the A subfiber of axonemes contains sites to which dynein preferentially attaches, MAPs may prevent recombination by interfering with cooperative binding to these specific sites. Dynein presumably binds with equal affinity to any protofilament on in vitro assembled microtubules, and, therefore, the MAPs may not be capable of effectively interfering with cooperative binding of dynein to these microtubules.
Adenosine Triphosphatases↗
Decoration of spindle microtubules with Dynein: evidence for uniform polarity.
Studies were conducted to determine whether the microtubules present within native spindles isolated from eggs of the surf clam, Spisula solidissima, could bind dynein obtained from axonemes of Tetrahymena thermophila. SDS gel electrophoresis revealed that the high molecular weight polypeptides that make up dynein cosedimented with the isolated spindles. Moreover, the ATPase activity of dynein bound to the spindle microtubules was stimulated approximately sevenfold. The birefringence retardation of spindles incubated without dynein decreased from 1.4 nm to an undetectable level within 45 min, whereas that of spindles incubated for the same period of time with dynein was 1.0 nm, approximately 70% of its initial value, thereby indicating that dynein stabilized spindle birefringence. Ultrastructural analysis revealed that each spindle microtubule was decorated with four to seven dynein arms attached by their "B" end, that which cross-bridges the B-subfiber within native axonemes. In addition, the polarity of the spindle microtubules could be determined by the orientation of the bound dynein arms. The results of these studies suggest that the half-spindle is composed of microtubules possessing the same polarity.
Adenosine Triphosphatases↗
Cilia, flagella, and microtubules.
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Animals↗
Dynein binds to and crossbridges cytoplasmic microtubules.
Dynein isolated from Chlamydomonas flagellar axonemes binds to microtubules assembled in vitro from 6S brain tubulin dimers. The dynein arms bind periodically along the length of the microtubules with a center-to-center spacing of 24 nm, equal to the periodicity of dynein arms on intact axonemes. The arms project from the in vitro assembled microtubules at an angle of approximately 55 degrees, thereby defining microtubule polarity. Dynein cosediments with microtubules through a sucrose gradient, as demonstrated by electron microscopy, gel electrophoresis, and ATPase analysis. In addition, dynein induces crossbridging between adjacent microtubules. Darkfield microscopy reveals that microtubules containing dynein are aggregated into large bundles; electron microscopy indicates that microtubules of the same polarity are crossbridged by a regular array of arms. Viewed by darkfield microscopy, addition of ATP to crossbridged microtubules causes their disaggregation; electron microscopy shows that the majority of these microtubules are no longer crossbridged. These observations are applicable to the determination of microtubule polarity and directionality of microtubule assembly in situ and suggest a role for dynein in cytoplasmic microtubule-based cellular movements.
Adenosine Triphosphatases↗
Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi.
Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.
Animals↗