The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1-3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays - termed an amphiaster ("a star on both sides" ) - that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) . Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ∼35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP - the small GTPase Ran in its GTP bound state - could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.
Bibliographical noteFunding Information:
We thank Jeff Salisbury for his generous gift of centrin 2 antibody, JoEllen Welsh and Holly Goodson for comments during the course of this work, Rick Miller for assistance in the initial fabrication of the microneedles, and Kul Karanjeet for technical assistance. J.E.H. was supported by training grants from the National Institutes of Health (T32 GM075762 and T32 CA080621). C.C.M. was supported by a graduate research fellowship from the National Science Foundation. S.L.S. was funded by a research grant from the National Science Foundation (NSF 0920555). K.T.V. and E.H.H. were supported by Research Scholar grants from the American Cancer Society. E.H.H. was supported by funds from the Hormel Institute, the Lyle Area Cancer Auction, and the Minnesota 5 th District Eagles Cancer Telethon. This work was supported by research grants from National Institute of General Medical Sciences to K.T.V. (GM060560) and E.H.H. (GM072754).