Biol. Bull. 207: 160. (October 2004)
© 2004 Marine Biological Laboratory
Characterization of Anastral, Bipolar Spindle Development and Atypical Cytokinesis in Ammonia-Activated Sea Urchin Eggs
John H. Henson1,2,
Jessica E. Davis2,
Charles B. Shuster1,3,
Christopher A. Fried1,2 and
Calvin R. Simerly4
1 Marine Biological Laboratory, Woods Hole, Massachusetts
2 Dickinson College, Carlisle, Pennsylvania
3 New Mexico State University, Las Cruces, New Mexico
4 Magee Women’s Research Institute, Pittsburgh, Pennsylvania
The mitotic apparatus (MA) of the early sea urchin embryo is often cited as the classic example of an aster-dominated MA that is organized via the introduction of a sperm-derived paternal centrosome. Previous studies have indicated that artificial activation of eggs can result in polymerization of microtubules associated with maternal centrosomal material, with the microtubule organization often taking the form of a monaster associated with condensed chromosomes. In the present study we have examined the generation of unusual bipolar spindle-like structures in eggs artificially activated with ammonia. Unfertilized sea urchin (Lytechinus pictus) eggs were activated by incubation in artificial seawater containing 15 mM ammonia chloride and fixed with cold methanol at the time appropriate for first division. The activated eggs were stained with antibodies against tubulin, actin, NuMA, centrosomal proteins, and the kinesin-like proteins Kif2, KRP110 and KRP170, and stained cells were viewed on a laser-scanning confocal microscope. The majority of activated eggs demonstrated poorly organized microtubules and/or the presence of monasters, but a subset contained atypical bipolar spindle-like structures. These spindles were small, anastral, capable of anaphase-like chromosome segregation, and overall very reminiscent of meiotic spindles. The spindle matrix protein NuMA, the centrosomal protein 4D2, and the spindle motor proteins Kif2, KRP110 and KRP170 all localized to these structures. The ammonia-induced spindles were also observed in living eggs using polarization optics on an LC-PolScope (courtesy of Rudolf Oldenbourg and Grant Harris of the Marine Biological Laboratory). The activated eggs went on to attempt cytokinesis, which often involved unusual linear arrays of microtubules in the region of the developing cleavage furrow. These results suggest that microtubule motor and matrix proteins have the ability to organize a meiotic-like spindle in sea urchin eggs in the absence of the paternal centrosome.
