Centrosome-independent mitotic spindle formation in vertebrates.
BACKGROUND
In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they 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-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy 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 microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however.


CONCLUSIONS
These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.
Khodjakov
A
A
Division of Molecular Medicine, New York State Department of Health, Department of Biomedical Sciences, Wadsworth Center, State University of New York, Albany, 12201-0509, 12222, USA. khodj@wadsworth.org.
Cole
R W
RW
Oakley
B R
BR
Rieder
C L
CL
D000818:Animals; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011418:Prophase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins