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© The Rockefeller University Press, 0021-9525/1997//1055 $5.00
The Journal of Cell Biology, Volume 138, Number 5, , 1997 1055-1066

Article

Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes



Tirso Gaglio, Mary A. Dionne, and Duane A. Compton

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.

1. Abbreviation used in this paper: NuMA, nuclear mitotic apparatus.

Please address all correspondence to Duane A. Compton, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel.: (603) 650-1990; Fax: (603) 650-1128; e-mail: duane.a.compton{at}dartmouth.edu

T. Gaglio dedicates this paper to his parents Evelio and Josefina. The authors would like to thank Dr. Tim Mitchison and his lab (University of California, San Francisco, CA) for being so generous with their Eg5 antibody, Dr. Yixian Zheng (Carnegie Institute of Washington, Wash. DC) for donating the {gamma}-tubulin antibody, and Dr. Trina Schroer (Johns Hopkins University, Baltimore, MD) for donating the dynactin-specific antibodies. We would also like to recognize the tolerance of Leslie Henderson and Bob Maue during microinjections and thank Alejandro Saredi, Vicki Mountain, Arijit Chakravarty, and Roger Sloboda for stimulating discussion during the preparation of this manuscript.


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