Centrosome-independent mitotic spindle formation in vertebrates

Background: In cells lacking centrosomes, the microtubule-organizing activi ty of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pat hway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labe ling with green fluorescent protein (GFP) and laser microsurgery we have be en able to selectively destroy centrosomes in living mammalian cells as the y enter mitosis, Results: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubul in-GFP. This feature allows us to use laser microsurgery to selectively des troy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that micr otubules form in acentrosomal cells randomly within the cytoplasm. When bot h centrosomes are destroyed during prophase these cells form a functional b ipolar spindle. Surprisingly, when just one centrosome is destroyed, bipola r spindles are also formed that contain one centrosomal and one acentrosoma l pole. Both the polar regions in these spindles are well focused and conta in the nuclear structural protein NuMA. The acentrosomal pole lacks pericen trin, gamma-tubulin, and centrioles, however. Conclusions: These results reveal, for the first time, that somatic cells c an use a centrosome-independent pathway for spindle formation that is norma lly masked by the presence of the centrosome. Furthermore, this mechanism i s strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.