THE DYNAMIC BEHAVIOR OF INDIVIDUAL MICROTUBULES ASSOCIATED WITH CHROMOSOMES IN-VITRO

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which, they are bound. The leng ths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-t ime assay that displays directly the movements of individual chromosom e-associated microtubules as they elongate and shorten. Chromosomes is olated from cultured Chinese hamster ovary cells were adhered to cover slips and then allowed to bind labeled microtubules. In the presence o f tubulin and GTP, these microtubules could grow at their chromosome-b ound ends, causing the labeled segments to move away from the chromoso mes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. T he link between a microtubule and a chromosome was mechanically strong ; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore-microtubule junc tion. The behavior of the microtubules in vitro was regulated by the c hromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of de polymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each, microtubule interacts with an increasing number of chromosome-associated binding sites as it appr oaches the kinetochore.