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© The Rockefeller University Press, 0021-9525/1998//1193 $5.00
The Journal of Cell Biology, Volume 141, Number 5, , 1998 1193-1205


Articles

Microinjection of Antibody to Mad2 Protein into Mammalian Cells in Mitosis Induces Premature Anaphase



Gary J. Gorbsky*, Rey-Huei Chen{ddagger}, and Andrew W. Murray§

* Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908; {ddagger} Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853; and § Department of Physiology, University of California, San Francisco, California 94143

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole–pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.


Abbreviations used in this paper: APC, anaphase-promoting complex; BUB, budding uninhibited by benzimidazole; MAD, mitotic arrest deficient.

G.J. Gorbsky was supported by a grant from the National Institute of General Medical Sciences.

Address all correspondence to Gary J. Gorbsky, Box 439 UVA Health Science Center, Charlottesville, VA 22908. Tel.: (804) 982-1654. Fax: (804) 982-3912. E-mail: gjg5y{at}virginia.edu



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