THE IMPACT OF CHROMOSOMES AND CENTROSOMES ON SPINDLE ASSEMBLY AS OBSERVED IN LIVING CELLS

We analyzed the role that chromosomes, kinetochores, and centrosomes p lay in spindle assembly in living grasshopper spermatocytes by reconst ructing spindles lacking certain components. We used video-enhanced, p olarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes a nd centrosomes make potent and very different contributions to the org anization of the spindle. Remarkably, the position of a single chromos ome can markedly affect the distribution of microtubules within a spin dle or even alter the fate of spindle assembly. In an experimentally c onstructed spindle having only one chromosome, moving the chromosome t o one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally . A spindle will also persist even when all chromosomes are detached a nd then removed from the cell. If, however, a single chromosome remain s in the cell but is detached from the spindle and kept in the cytopla sm, the spindle disassembles. One might expect the effect of chromosom es on spindle assembly to relate to a property of a specific site on e ach chromosome, perhaps the kinetochore. We have ruled out that possib ility by showing that it is the size of chromosomes rather than the nu mber of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosome s. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.