Dynein binds to and crossbridges cytoplasmic microtubules

Abstract

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.