A MODEL FOR THE PROPOSED ROLES OF DIFFERENT MICROTUBULE-BASED MOTOR PROTEINS IN ESTABLISHING SPINDLE BIPOLARITY

Background: In eukaryotes, assembly of the mitotic spindle requires th e interaction of chromosomes with microtubules. During this process, s everal motor proteins that move along microtubules promote formation o f a bipolar microtubule array, but the precise mechanism is unclear. I n order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles a ssemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly i n the absence of paired cues, such as centrosomes and kinetochores, wh ose microtubule-organizing properties might mask the action of motor p roteins. Results: We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xeno pus kinesin-like protein 1 (Xklp1) led either to the dissociation of c hromatin beads from microtubule arrays, or to collapsed microtubule bu ndles on beads. Inhibition of Eg5 resulted in monopolar microtubule ar rays emanating from chromatin beads. Addition of antibodies against dy nein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxyterminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhi bition of XCTK2 and dynein dramatically increased the severity of spin dle pole defects. Inhibition of XklpP caused only minor spindle pole d efects. Conclusions: Multiple microtubule-based motor activities are r equired for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor pr oteins in this process. (C) Current Biology Publications ISSN 0960-982 2.